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QP51 9  .M31 2  Manual  for  the  physi 

RECAP 


MANUAL 


FOR  THE 


PHYSIOLOGICAL  CHEMICAL 
LABORATORY 


JOHN  A.  MANDEL 


QPS~iQ  M3IZ 

Columbia  tBntoerafap 
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College  of  $tjpgtctansi  anb  burgeon* 
Htbrarp 


Gift  of 
Dr.  Jerome  P  Wet  s~ber 


MANUAL 


FOR   THE 

PHYSIOLOGICAL   CHEMICAL 
LABORATORY. 

FOR   THE    USE    OF    MEDICAL 
STUDENTS. 


JOHN  A.  MANDEL, 

Adjunct  Professor  of  Physiological  Chemistry  at  the 
Bellevue  Hospital  Medical  College. 


NEW  YORK. 

JOS.   V.  STANDISH. 

1897. 


/vv 


Ube  1knicf?ei'bocher  press  IRew  lOorf? 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 
Columbia  University  Libraries 


http://www.archive.org/details/manualforphysiolOOmand 


Manual  for  the  Physiological 
Chemical  Laboratory. 


Lesson  I. 

CARBOHYDRATES. 

A.compound  containing  C,  H,  and  O,  where  the  H 
and  O  are  in  the  proportion  to  form  water. 
Three  groups. 

MONOSACCHARIDES.  DISACCHARIDES.  POr.YSACCHARIDKS. 

C6H12Oc.  C1SHSS01.1.  C6H10O5. 

Dextrose,  glucose,  Cane  sugar,  Starch, 

Laevulose,  Lactose,  Dextrin, 

Inosite,  Maltose,  Glycogen, 

Galactose,  Cellulose. 

DEXTROSE,  GLUCOSE,  GRAPE,  SUGAR,  ETC. 

The  most  important  member  of  the  first  group  is 
dextrose,  which  occurs  in  certain  fruits  and  grains,  and 
in  the  blood,  lymph,  chyle,  diabetic  urine,  etc. 

Experiment  i.  Place  some  in  a  test  tube  with  water. 
It  dissolves.     It  has  a  sweetish  taste. 

Experiment  2.  Fill  a  test  tube  one  quarter  full  with 
silver  nitrate  (AgN03)  solution,  and  add  a  few  drops 

1 


2  LESSON  I. 

of  ammonium  hydrate  (NH4OH)  until  the  precipitate 
formed  just  dissolves.  Add  a  few  drops  of  the  above 
dextrose  solution  to  this,  and  warm  in  water-bath.  A 
deposit  of  a  metallic  silver  mirror  is  obtained,  showing 
that  dextrose  has  a  reducing  action  on  certain  metallic 
solutions  when  in  an  alkaline  condition,  and  most  of 
the  chemical  tests  for  dextrose  depend  on  this  prop- 
erty. 

Experiment  j.  Trommer  s  test.  Place  a  few  drops 
copper  sulphate  (CuS04)  in  a  test  tube,  and  an  equal 
amount  of  water,  and  then  caustic  soda  (NaOH)  until 
a  little  of  the  copper  hydrate  formed  remains  undis- 
solved. Add  to  this  a  drop  or  two  of  the  dextrose 
solution.  The  copper  hydrate  dissolves.  Apply  gentle 
heat.  A  yellow  reduction  of  hydrated  sub-oxide  of 
copper,  and  then  red  sub-oxide  of  copper  (Cu30),  sepa- 
rates even  below  the  boiling  point. 

Care  must  be  taken  not  to  have  an  excess  of  either 
copper  sulphate  or  caustic  soda  in  this  test,  otherwise 
interferences  are  liable  to  occur. 

Experiment  4..  Moore's  test.  Make  some  of  the  dex- 
trose solution  alkaline  with  caustic  soda,  and  apply 
heat.  The  solution  turns  yellow,  red,  brown,  or  black, 
depending  upon  the  amount  of  dextrose  in  the  solu- 
tion. Add  a  few  drops  of  nitric  acid  (HNOs)  to  the 
solution.  The  color  disappears,  and  an  odor  of  caramel 
or  burnt  sugar  is  given  off. 

Experiment  5.  Fehlings  test.  Place  some  copper 
sulphate  in  a  test  tube,  and  an  equal  amount  of  caustic 
soda  solution,  then  a  solution  of  Rochelle  salt  (sodium- 
potassium  tartrate  NaKC4H4Oe,  4.H30)  until  the  pre- 
cipitate is  dissolved,  and  a  clear,  deep  blue  solution  is 


CARBOHYDRATES.  3 

obtained.  This  constitutes  Fehling's  solution.  Heat 
the  upper  part  of  the  liquid  to  the  boiling  point  by 
holding  the  test  tube  at  the  lower  end  and  applying 
the  heat  above.  No  change,  should  occur.  Add  a 
drop  of  the  dextrose  solution.  Immediate  reduction 
of  the  yellow  hydrated  sub-oxide  of  copper,  and  then  a 
red  sub-oxide  of  copper. 

Experiment  6.  Barfoed's  test.  Add  some  of  Bar- 
foed's  solution  (a  solution  of  copper  acetate  with  acetic 
acid)  to  some  of  the  dextrose  solution  in  a  test  tube, 
and  apply  heat.  A  reduction  of  copper  sub-oxide  is 
produced.  This  test  distinguishes  dextrose  from  lac- 
tose and  maltose. 

Experiment  y.  Boettgers  test.  Fill  a  test  tube  one 
half  full  of  water,  add  a  little  of  the  dextrose  solution 
and  some  solid  bismuth  sub-nitrate,  and  now  make  al- 
kaline with  sodium  carbonate  (Na2COs).  Heat  to 
boiling.  The  white  bismuth  sub-nitrate  will  turn  gray, 
brown,  or  black,  depending  on  the  amount  of  dextrose 
in  the  solution,  and  will  collect  at  the  bottom  of  the 
test  tube. 

Experiment  8.  Place  some  of  the  dextrose  solution 
in  the  saccharometer,  and  add  a  small  piece  of  yeast 
thereto.  Place  in  a  warm  locality  at  a  temperature  of 
about  400  C.  Bubbles  of  gas  collect  after  a  time. 
This  is  called  fermentation,  and  the  gas  which  collects 
is  carbon  dioxide  (C02),  while  another  body,  alcohol 
(C2H3OH),  remains  in  the  liquid.  C6H1206  = 
2C2-H5OH  +  2C02. 

CANE   SUGAR. 
Occurs   in   the  juices  of   many  plants,   in   fruits,  in 
flowers,  and  in  honey;   does  riot  occur  in  the  animal 


4  LESSON  I. 

body,   but   undergoes  transformations  when   partaken 
of. 

Experiment  p.  Place  some  cane  sugar  in  some  water 
in  a  test  tube.  It  dissolves  readily,  forming  a  solution 
having  a  sweetish  taste. 

Experiment  10.  Repeat  Experiment  5,  using  a  solu- 
tion of  cane  sugar  instead  of  dextrose.  No  reduction 
on  the  application  of  heat. 

Experiment  11.  Fill  a  test  tube  one  half  full  with 
the  cane  sugar  solution,  acidify  with  a  drop  or  two  of 
sulphuric  acid  (H3S04),  and  boil  for  a  few  minutes. 
This  operation  will  convert  the  cane  sugar  into  a  mix- 
ture of  dextrose  and  laevulose,  called  invert  sugar, 
C12H33On  +  H30  =  C6H1306  +  C6H1306. 

This  process  is  similar  to  the  action  of  the  invertin 
of  the  intestinal  juice  on  cane  sugar  in  the  intestin. 

Experiment  12.  Neutralize  this  solution  with  caustic 
soda  (NaOH),  and  repeat  Experiment  5.  A  reduction 
of  the  red  sub-oxide  is  obtained,  showing  the  presence 
of  dextrose  and  laevulose. 

Cane  sugar  does  not  ferment  under  the  action  of 
yeast,  but  readily  undergoes  lactic  acid  fermentation 
under  the  action  of  certain  ferments. 

LACTOSE   OR   MILK   SUGAR. 

Important  constituent  of  the  milk  of  mammals  and 
is  sometimes  found  in  the  urine  of  women  in  the  early 
days  of  lactation,  or  after  weaning. 

Experiment  ij.  Place  some  of  the  lactose  in  water 
in  a  test  tube.  It  does  not  dissolve  very  readily  and 
has  a  feeble  sweet  taste. 

Experiment  14..     Repeat  Experiment  5,  using  a  solu- 


CA  A' BOH  YDKA  TES.  5 

tion  of  lactose.     A  reduction  similar  to  that  obtained 
with  dextrose  is  the  result. 

Experiment  ij.  Add  some  Kephir  ferment  to  some 
lactose  solution  and  keep  warm  for  a  short  time,  the 
solution  turns  acid  as  shown  by  placing  a  drop  of  the 
solution  on  a  piece  of  blue  litmus  paper.  This  acid  is 
lactic  acid  Cl2Y[22Olx  -f~H20  =  4C3H603.. 

Lactose.  Lactic  acid. 

MALTOSE   OR   MALT   SUGAR. 

Formed  by  the  action  of  malt  diastase  on  starch.  Is 
the  chief  product  of  the  action  of  saliva  and  pancreatic 
juice  on  starch  during  digestion. 

Experiment  16.  Dissolve  some  in  water  and  observe 
the  sweet  taste  of  the  solution. 

Experiment  ij.  Repeat  Experiment  5,  using  the 
above  solution  of  maltose.  A  reduction  is  obtained 
but  not  so  pronounced  as  with  dextrose. 

Experiment  iS.  Apply  Barfoed's  test  (see  Experi- 
ment 6)  to  the  maltose  solution.     No  reduction. 

It  really  undergoes  fermentation  by  yeast,  and  when 
heated  with  very  dilute  sulphuric  acid  it  yields  dex- 
trose.    Diastatic  enzymes  have  a  similar  action. 

STARCH. 

Found  in  nearly  all  plants.  Is  most  abundant  in  the 
cereals.  Occurs  as  microscopic  granules  of  various 
sizes  and  structure.  Does  not  occur  in  the  animal 
body  except  as  food. 

Experiment  ig.  Place  some  in  water.  Does  not 
dissolve  but  disintegrates  yielding  a  white  mud. 

Experiment  20.     Boil  some  water  in  a  test  tube  and 


6  LESSON  I. 

add  some  of  the  above  mud  thereto.  An  opalescent 
solution  or  jelly-like  mass  is  the  result,  depending  upon 
the  amount  of  starch  added. 

Experiment  21.  Place  a  little  starch  solution  in  a 
test  tube  and  dilute  with  water.  Add  a  drop  of  tinct- 
ure of  iodine  thereto.  Deep  blue  coloration  is  obtained, 
which  disappears  on  heating  and  reappears  on  cooling 
again. 

Experiment  22.  Boil  some  starch  solution  with  some 
dilute  hydrochloric  acid  (HO)  for  some  time,  neutral- 
ize with  caustic  soda  (NaOH)  and  repeat  Experiment 
5,  using  a  portion. 

A  reduction   is  obtained  showing  the  presence   of 
dextrose.     The  action  of  the  acid  is  expressed  by  the 
following  equation  : 
3C6H10O5+H3O  =  C6H12O6+C6H10O5+C6H10Og 

Starch.  Dextrose.         Achroo-dextrin.      Erythro-dextrin. 

Experiment  2j.  Moderately  heat  some  of  the  starch 
solution  with  an  infusion  of  malt  (containing  diastase) 
for  some  time.  It  first  becomes  clear  due  to  the  for- 
mation of  soluble  starch.  Apply  Fehling's  test  (Ex- 
periment 5)  to  the  product.  A  reduction  is  obtained 
due  to  the  presence  of  maltose.  The  change  is  ex- 
pressed by  the  following  equation  : 
ioC6H10O5  +4H30  =  4C12H33Otl  +2  C6H10O5. 

Starch.  Maltose.  Dextrin. 

The  action  of  the  ptyalin  of  the  saliva  and  amylopsin 
of  the  pancreatic  juice  on  starch  is  also  represented  by 
the  above  change. 

GLYCOGEN   OR   LIVER   STARCH. 
Occurs  chiefly  in  the  liver,  muscles,  and  nearly  all 
tissues  of  the  animal  body,  and  is  a  regular  constituent 
of  all  developing  animal  cells. 


CAR  BOH  YDRA  TE  S.  J 

Experiment  2cf..  Place  a  little  in  some  water  in  a  test 
tube.  It  dissolves  yielding  an  opalescent  solution 
having  no  marked  taste. 

Experiment  25.  Add  a  drop  of  tincture  of  iodine  to 
a  portion  of  the  above  solution.  A  wine  red  coloration 
is  obtained,  which  disappears  on  heating. 

Experiment  26.  Boil  the  other  portion  of  the  gly- 
cogen solution  with  some  dilute  hydrochloric  acid 
(HO),  neutralize  with  caustic  soda,  and  then  test  for 
dextrose  by  repeating  Experiment  5.  The  boiling  with 
the  acid  has  converted  the  glycogen  into  dextrose  and 
dextrin,  similar  to  Experiment  22.  Certain  ferments 
such  as  that  of  the  liver  change  glycogen  into  dextrose 
and  dextrin. 

DEXTRINS. 

Found  in  the  blood  and  muscles  of  animals  to  a 
slight  extent.  They  are  products  of  the  diastatic  fer- 
ments on  starch,  etc.  Two  varieties — Achroo-dextrin 
and  Erythro-dextrin.  Common  dextrin  is  chiefly  Ery- 
thro-dextrin. 

Experiment  2j.  Place  some  in  water  in  a  test  tube. 
It  dissolves  and  a  strong  solution  forms  a  gummy  and 
sticky  mass. 

Experiment  28.  Add  some  tincture  of  iodine  to 
the  dextrin  solution.     Red  coloration. 

Experiment  29.  Boil  some  of  the  dextrin  solution 
with  dilute  hydrochloric  acid  (HC1)  for  a  few  minutes. 
Neutralize  with  caustic  soda  (NaOH)  and  test  for  dex- 
trose by  Fehling's  solution  (Experiment  5).  The  acid 
has  converted  the  dextrin  into  dextrose  as  shown  by 
the  reduction. 


Lesson  II. 

FATS. 

Almost  all  fats  and  oils  are  compound  ethers  of 
glyceryl,  C3  H5",  or  triglycerides. 

The  most  important  fats  are  tri-stearine  C3  H5 
(C18  H35  03)3,  tri-palmitine  C3  H5  (C16  H31  03)3, 
and  tri-olein  C3  Hg  (C18  H33  Os)3.  These  are  com- 
pound ethers  of  stearic,  palmitic,  and  oleic  acids  with 
glyceryl. 

Experiment  i.  Place  some  of  the  fat  in  water  in  a 
test  tube.  It  does  not  dissolve  but  floats  on  the  liquid, 
hence  it  is  lighter  than  water. 

Experiment  2.  Apply  heat  to  the  water  and  ob- 
serve that  the  fat  melts  much  before  the  water  begins 
to  boil. 

Experiment  j.  Rub  a  little  of  the  fat  on  a  piece  of 
paper  and  observe  that  it  makes  a  transparent  spot. 

Experiment  ./.  Heat  a  little  of  the  fat  on  foil  with 
some  potassium  bisulphate  (KHS04)  and  note  that  it 
melts  readily,  flowing  about  the  foil  and  giving  off  a 
marked  and  characteristic  irritating  odor  of  acrolein. 

It  inflames,  burning  with  a  sooty  flame. 

Experiment  5.  Shake  some  of  the  liquid  fat  given 
with  water  in  a  test  tube.  The  oil  globules  readily 
conglomerate  and  rise  to  the  surface. 

s 


FA  TS.  9 

Experiment  6.  Shake  another  portion  with  an  albu- 
min solution.  The  oil  globules  do  not  readily  con- 
glomerate, but  forma  milky  white  emulsion. 

Experiment  7.  Shake  a  third  portion  of  the  liquid 
fat  with  water  containing  a  little  soap  solution.  An 
immediate  and  permanent  emulsion  is  obtained  on 
slightly  shaking. 

These  facts  are  of  the  greatest  importance  in  the 
digestion  and  assimilation  of  fats  in  the  intestinal 
tract. 

Experiment  S.  Add  an  alcoholic  solution  of  caustic 
soda  to  some  of  the  solid  fat  and  heat  on  the  water- 
bath  for  some  time.  The  product,  a  soap,  is  character- 
ized by  its  forming  a  lather  if  shaken  with  water.  This 
process  is  called  saponification,  and  may  be  expressed 
by  the  following  equation  : 

C3Hs(C18H35Oa)3+3NaOH=3NaC18H3503+ 

Tri-stearin.  Caustic  soda.  Sod.  stearate  or  soap. 

C3H803. 

Glycerin. 

Experiment  g.  Treat  some  of  the  soap  solution  with 
some  dilute  sulphuric  acid  (H2  S04).  A  decomposi- 
tion with  the  precipitation  of  the  insoluble  fatty  acids 
is  the  result. 

2NaC18H3503-fH3S04  =  2C18H360, +Na2S04. 

Soap.  Sulphuric  acid.  Stearic  acid.  Sod.  sulphate. 

Experiment  10.  Treat  a  small  quantity  of  the  liquid 
fat  with  some  pancreatin  and  add  a  small  amount  of 
sodium  carbonate  (Na2C03).  Heat  in  the  water-bath 
at  400  C.  for  some  time.  The  fat-splitting  ferment  of 
the  pancreas  will  decompose  a  part  of  the  fat  into  fatty 


10  LESSON  II. 

acids  and  glycerine  which  combine  with  the  sodium 
carbonate  forming  soaps  which  in  turn  cause  the 
unacted  on  fats  to  form  an  emulsion  on  shaking. 

C3H5(Ci8H3502N>3+3H20  =  3C18H36024-C3H8C)3. 

Tri-stearin  Stearic  acid.  Glycerin. 

Experiment  n.  Shake  some  water  containing  a 
little  oil  with  ether.  The  ether  dissolves  the  fat 
readily.  Decant  the  ethereal  solution  into  a  watch 
glass,  and  allow  the  ether  to  evaporate,  leaving  the  fat. 

Experiment  12.  Apply  a  little  of  the  fat  to  a  piece 
of  blue  litmus  paper.     No  reaction. 

Experiment  ij.  Apply  a  little  of  the  rancid  fat  to  a 
piece  of  blue  litmus  paper.  A  marked  acid  reaction  is 
obtained,  showing  that  the  rancidity  of  fats  is  due  to 
free  fatty  acids. 

The  free  fatty  acids  form  soaps  directly,  without  de- 
composition, with  sodium  carbonate  (Na3C03)  with 
the  disengagement  of  carbon  dioxide  (C03)gas.  They 
do  not  evolve  an  odor  of  acrolein  when  heated  with 
potassium  bisulphate.  The  fatty  acids  have  an  acid 
reaction.  These  properties  distinguish  them  from  the 
neutral  fats. 


Lesson  III. 
PROTEIN  SUBSTANCES. 

The  most  important  class  of  substances  that  occur 
in  artimal  or  vegetable  organisms  and  necessary  for 
the  phenomena  of  life.  They  are  highly  complex  in 
their  constitution  and  contain  C,H,0,N,S,  and  some  P. 
The  nitrogen  is  the  most  important  element. 

The  protein  substances  are  divided  into  various 
groups  having  different  properties  and  characteristics. 
They  are  as  follows:  albumins,  globulins,  nucleo-albu- 
mins,  albuminates,  albumoses  and  peptones,  coagulated 
proteids,  haemoglobin,  glyco-proteids,  nucleo-proteids, 
keratin,  elastin,  collagen,  etc. 

Experiment  i.  Place  a  little  of  the  albumin  on  foil 
and  apply  heat.  It  blackens,  burns  with  a  character- 
istic odor  of  burnt  hair  or  horn,  and  leaves  no  residue. 
This  odor  is  characteristic  of  nitrogenous  organic  com- 
pounds. 

Experiment  2.  Place  some  in  water  in  a  test  tube. 
It  dissolves,  but  not  readily,  yielding  an  opalescent 
solution. 

Experiment  3.  Apply  heat  to  some  albumin  solution 
in  a  test  tube.  The  solution  becomes  cloudy  and  fin- 
ally a  coagulum  forms.  This  is  called  heat  coagula- 
tion. Add  a  drop  of  acetic  acid  to  the  above  and  the 
coagulum  will  be  increased. 


12  LESSON  III. 

Experiment  </.  Saturate  some  albumin  solution  with 
ammonium  sulphate  (NH"4)3S04.  A  precipitate  of 
albumin  is  produced.  All  protein  substances  with  the 
exception  of  peptones  are  precipitated  by  saturation 
with  this  salt.  This  is  a  means  of  separating  peptones 
from  other  protein  substances. 

Experiment  5.  Place  a  solution  of  albumin  contain- 
ing some  sodium  chloride  (NaCl)  in  a  parchment  dialy- 
ser  as  described,  and  place  it  in  a  vessel  of  water.  It 
will  be  found  that  the  water  outside  will  acquire  a  salty 
taste  after  a  time,  because  the  salt  has  passed  through 
the  membrane.  The  presence  of  the  salt  may  be  tested 
by  placing  some  of  the  water  in  a  test  tube  and  adding 
some  silver  nitrate  (AgNOs),  which  will  give  a  white 
precipitate.  No  albumin  will  be  found  in  the  water  on 
the  outside. 

This  process  is  called  dialysis  or  diffusion  through 
membranes.  The  salts  are  diffusible  and  are  called 
crystalloid  substances,  while  the  protein  bodies,  with 
the  exception  of  peptones,  do  not,  or  with  difficulty, 
diffuse,  and  are  called  colloid  substances.  Dialysis  is 
made  use  of  in  separating  colloid  from  crystalloid 
bodies. 

Experiment  6.  Color  reactions.  Add  some  Millon's 
reagent  to  a  little  of  the  albumin  solution.  A  precipi- 
tate is  formed,  which  slowly  at  the  ordinary  tempera- 
ture, but  quickly  at  the  boiling  point,  turns  red, 
depending  upon  the  amount  of  albumin  present. 

The  solid  proteids  give  the  same  reaction. 

Millon's  reagent  consists  of  a  solution  of  mercuric 
nitrate  in  nitric  acid  containing  some  nitrous  acid. 

Experiment  7.     Xantho-proteic  reaction.     Add  a  drop 


PROTEIN  SUBSTANCES.  I  3 

of  nitric  acid  (HN03)  to  some  solid  albumin  on 
a  watch  glass.  It  turns  yellow  and  orange-yellow. 
After  a  little  while  add  a  few  drops  of  ammonium 
hydrate  (NH4OH)  or  caustic  soda  (NaOH)  when  the 
spot  becomes  red. 

Experiment  8.  Biuret  reaction.  Place  one  or  two 
drops  dilute  copper  sulphate  (CuS04)  in  a  test  tube 
and  add  an  excess  of  caustic  soda  (NaOH).  Add  some 
albumin  solution  to  this  and  apply  heat.  A  violet 
color  is  produced  which  deepens  in  tint  on  boiling.  A 
reduction  of  copper  sub-oxide  may  take  place. 

Experiment  p.  Add  two  or  three  drops  nitric  acid 
(HNO,)  to  some  dilute  albumin  solution.  A  precipi- 
tate occurs  which  re-dissolves  on  adding  an  excess  of 
the  acid. 

Experiment  10.  Place  some  nitric  acid  (HN03)  in  a 
test  tube  and  allow  the  filtered  albumin  solution  to 
flow  down  gently  on  its  surface.  A  well-defined  ring 
or  zone  of  precipitated  albumin  appears  at  the  juncture 
of  the  two  liquids.  Most  mineral  acids  act  in  the  same 
manner. 

Experiment  II.  Add  some  mercuric  chloride  (HgCl2) 
to  some  albumin  solution.  A  precipitate  of  albumin  is 
obtained.  Most  metallic  solutions  have  the  same  action, 
hence  the  use  of  albumin  as  an  antidote  in  cases  of 
metallic  poisoning. 

Experiment  12.  Acidify  some  albumin  solution  with 
acetic  acid  (C2H402)  and  add  a  few  drops  of  potassium 
ferrocyanide  (K4Fe(CN)6)  solution.  A  white  precipi- 
tate of  albumin  is  obtained. 

Experime?it  ij.  Acidify  some  albumin  solution 
with    acetic    acid    and    add    a    few    drops    picric    acid 


14  LESSON  III. 

(C6H2(NO)3OH)  solution.  A  white  precipitate  of 
albumin  is  produced. 

Experiment  14.  Add  some  alcohol  to  some  albumin 
solution  in  a  test  tube.  The  albumin  will  be  precipi- 
tated. 

Experiment  ij.  Place  a  little  dilute  albumin  solution 
in  a  test  tube,  add  some  pepsin  and  one  drop  hydro- 
chloric acid,  and  place  in  the  water  bath  at  about  400 
C.  for  fifteen  minutes. 

The  pepsin  will  convert  the  albumin  into  albumoses 
and  peptones,  which  are  the  chief  products  of  the  pro- 
teolytic ferments,  such  as  the  pepsin  of  the  gastric 
juice  and  the  trypsin  of  the  pancreatic  juice. 

Acidify  the  solution  with  a  few  drops  of  acetic  acid 
and  heat  to  the  boiling  point.  This  precipitates  the 
unconverted  albumin,  which  may  be  removed  by  filtra- 
tion and  the  presence  of  albumoses  and  peptones  deter- 
mined by  applying  the  Biuret  test.  (See  Experiment 
8,  which  gives  a  rose-red  color.) 

Experiment  16.  Repeat  Experiment  15,  but  instead 
of  using  pepsin  use  pancreatin  and  show  that  the  pro- 
ducts are  about  the  same  in  both  cases. 

In  this  experiment  do  not  make  the  solution  acid 
with  hydrochloric  acid,  but  add  one  drop  of  sodium 
carbonate  solution  instead. 

Experiment  iy.  Add  some  peptone  to  some  water 
in  a  test  tube.     It  dissolves  readily. 

Experiment  18.  Apply  heat  to  a  portion  of  this  solu- 
tion. No  coagulation  showing  a  difference  from  other 
protein  substances. 

Experiment  19.  Add  some  nitric  acid  to  some  of  the 
peptone  solution.     No  precipitation. 


PROTEIN  SUBSTANCES.  I  5 

Experiment  20.  Apply  the  Biuret  test  to  some  pep- 
tone solution.  A  rose-red  color  is  obtained  instead  of 
a  violet  color,  as  given  by  other  protein  substances. 

Experiment  21.  Place  a  peptone  solution  in  a  parch- 
ment dialyser  and  place  it  in  a  vessel  of  pure  water. 
After  a  time  the  peptones  will  have  diffused  through 
the  membrane,  as  shown  by  applying  the  Biuret  test 
to  the  water  on  the  outside. 


Lesson  IV. 

DIGESTION. 

Is  to  separate  those  constituents  of  the  food  which 
serve  as  the  nutriment  of  the  body  from  those  which 
constitute  the  waste  and  to  separate  each  in  such  a 
form  that  it  may  be  easily  taken  up  by  the  blood  from 
the  alimentary  canal. 

SALIVARY  DIGESTION. 

Experiment  i.  Collect  as  much  saliva  in  a  test  tube 
as  possible. 

Experiment  2.  Apply  a  little  to  a  piece  of  red  litmus 
paper.  It  turns  blue,  showing  it  to  be  alkaline.  Di- 
rectly after  a  meal  it  may  be  acid. 

Experiment  j.  Add  a  drop  of  hydrochloric  acid 
(HC1)  to  some  saliva  in  a  test  tube  and  then  a  few 
drops  of  ferric  chloride  (Fe2CL6).  A  blood-red  color- 
ation denotes  the  presence  of  sulphocyanides.  The 
depth  of  color  varies  with  different  saliva,  because  the 
amount  of  sulphocyanides  varies  very  markedly. 

Experiment  /f..  Make  some  dilute  starch  solution 
(as  described  in  Lesson  I.,  Experiment  20)  and  add 
thereto  some  fresh  saliva  and  keep  in  the  water-bath 
at  a  temperature  of  50°-55°  C.  for  some  little  time. 
Observe  that  the  starch  solution  becomes  clear.  Test 
a  portion  of  the  solution  with  tincture  of  iodine  when, 

16 


DIGESTION.  17 

instead  of  a  blue  reaction   for  starch,  a  red  coloration 
due  to  dextrin  will  be  obtained. 

Test  the  presence  of  sugar  (maltose)  in  the  liquid  by- 
means  of  Fehling's  or  Boettger's  test.     (See  Lesson  I.) 

GASTRIC  DIGESTION. 

Experiment  j.  Test  the  reaction  of  the  artificial 
gastric  juice  given  by  means  of  a  piece  of  blue  litmus 
paper.     It  is  found  to  be  acid. 

Experiment  6.  Add  a  drop  of  silver  nitrate  to  a  little 
of  the  gastric  juice.  A  white  precipitate  of  silver 
chloride  (AgCl)  shows  the  presence  of  hydrochloric 
acid  (HC1).  This  precipitate  is  soluble  in  ammonium 
hydrate  (NH4OH). 

Experiment  7.  Place  a  drop  of  the  gastric  juice  on  a 
piece  of  Congo  red  paper.  It  turns  blue,  due  to  the 
free  hydrochloric  acid. 

Experiment  8.  Spread  a  few  drops  of  Gunsburg  s 
solution  (consisting  of  an  alcoholic  solution  of  phloro- 
glucin-vanillin)  out  in  a  thin  layer  upon  a  watch  glass, 
or  porcelain  dish,  and  then  gently  warm  ;  then  a  drop 
of  the  gastric  juice  is  allowed  to  flow  across  it,  or  a 
glass  rod  dipped  in  the  solution  is  drawn  across  the 
plate.  If  free  hydrochloric  acid  be  present,  a  deep 
scarlet-red  streak  is  developed. 

Experiment  g.  Test  for  lactic  acid.  Add  a  few 
drops  of  the  gastric  juice  to  some  Uffelmann's  solu- 
tion. This  solution  is  made  by  mixing  a  few  drops 
neutral  ferric  chloride  (Fe2Cl6)  solution  with  2  drops 
of  pure  carbolic  acid  (C6H5OH),  and  adding  water 
until  the  solution  has  a  beautiful  amethyst-blue  color. 


1 8  LESSON  IV. 

In  the  presence  of  lactic  acid  the  blue  color  is  changed 
to  yellow  instantly. 

Experiment  10.  Place  a  piece  of  hard-boiled  egg  in 
a  test  tube  and  add  a  little  of  the  gastric  juice.  Keep 
this  at  a  temperature  of  about  400  C,  and  observe  the 
swelling  up  of  the  edges,  the  corrosion  and  the  gradual 
disappearance  or  solution  of  the  albumin  being  con- 
verted into  albumoses  and  peptones. 

Experiment  11.  Test  the  presence  of  peptones  in 
the  solution  by  means  of  the  Biuret  test.  (See  Lesson 
3,  Experiment  20.) 

Experiment  12.  Warm  a  little  milk  to  about  400  C. 
and  then  add  some  of  the  neutral  gastric  juice.  An 
immediate  coagulation  of  the  milk  is  observed,  due  to 
the  enzyme  called  Rennin  in  the  gastric  juice. 

PANCREATIC  DIGESTION. 

Experiment  ij.  Apply  some  of  the  pancreatic  juice 
to  a  piece  of  red  litmus  paper.  It  turns  it  blue,  show- 
ing it  to  be  alkaline. 

Experiment  14..  Make  some  dilute  starch  solution 
and  add  some  pancreatic  juice  thereto,  and  keep  at  a 
temperature  of  about  400  C.  for  some  little  time.  Ob- 
serve that  the  starch  solution  becomes  clear  and  the 
presence  of  sugar  (maltose)  may  be  shown  by  applying 
Fehling's  test  to  the  solution. 

The  amylopsin  of  the  pancreatic  juice  converts  starch, 
etc.,  into  maltose  and  dextrin. 

Experiment  15.  Place  some  liquid  fat  with  some 
water  in  a  test  tube  and  add  a  little  pancreatic  juice  ; 
keep  at  a  temperature   of  400  C.  for  some  time.     A 


DIGESTION.  19 

perfect  emulsion  of  the  fat  will  be  obtained  if  the  con- 
tents of  the  tube  is  shaken.  (See  Experiment  10, 
Lesson  2.) 

Experiment  16.  Place  a  piece  of  hard-boiled  egg  in 
a  test  tube  and  add  some  of  the  pancreatic  juice  and 
keep  at  400  C.  for  some  time.  Observe  the  action  on 
the  albumin  and  the  gradual  solution  of  the  piece. 

The  trypsin  converts  the  albumin  into  albumoses 
and  peptones  which  may  be  tested  for  by  the  Biuret 
test.    (See  Lesson  3,  Experiment  20.) 


Lesson  V. 
BLOOD  AND  BILE. 

BLOOD. 

Observe  the  obtainment  of  salt  plasma  from  the 
blood  of  a  dog. 

Also  the  collection  of  blood  in  a  vessel  surrounded 
with  an  ice  mixture. 

Observe  the  coagulation  of  the  salt  plasma  on  the 
addition  of  water. 

Also  the  coagulation  of  the  cold  blood  on  allowing 
it  to  attain  the  temperature  of  the  room. 

Allow  this  last  to  stand  quietly,  so  that  the  clot  may- 
settle,  leaving  the  clear  serum  above. 

Experiment  i.  Place  a  drop  of  the  serum  on  a  piece 
of  red  litmus  paper.  It  turns  blue,  showing  the  blood 
serum  to  be  alkaline. 

Experiment  2.  Saturate  some  of  the  serum  with 
magnesium  sulphate  (MgS04).  A  precipitate  of  ser- 
globulin  is  produced.  Filter  this  precipitate  off,  and 
keep  the  nitrate. 

Experiment  j.  Remove  some  of  the  substance  col- 
lected on  the  filter,  and  place  it  in  a  test  tube  with 
water. 

It  dissolves,  due  to  the  salt  retained  by  the  precipi- 


BLOOD  AND  BILE.  21 

tate.  Pure  ser-globulin  is  insoluble  in  water,  but  dis- 
solves in  dilute  salt  solutions. 

Experiment  7.  Heat  a  portion  of  the  above  ser- 
globulin  solution.     It  coagulates. 

Experiment  3.  Apply  the  Biuret  test  (see  Experi- 
ment 8,  Lesson  3)  to  another  portion.  A  violet  reac- 
tion will  be  obtained. 

Experiment  6.  Add  some  sodium  sulphate  (Na2S04) 
to  the  cold  filtrate  obtained  in  Experiment  2.  A 
precipitate  of  ser-albumin  occurs.  Collect  the  preci- 
pitate on  a  filter. 

Experiment  7.  Remove  the  precipitate  from  the 
filter,  and  place  it  in  a  test  tube  with  water.  It  dis- 
solves. 

Heat  a  portion,  and  observe  that  it  coagulates. 

Apply  Experiments  9,  10,  12,  and  13,  Lesson  3,  to 
some  of  the  solution.  The  relationship  of  ser-globulin 
to  ser-albumin  in  human  blood  serum  is  1:1.5,  m  oxen, 
1:0.842,  in  dogs,  1:1.8. 

Experiment  8.  Wash  some  of  the  blood  clot  in 
water  until  nearly  white  and  free  from  blood  cor- 
puscles. 

Observe  the  thready  or  fibrous  character  of  the 
fibrin. 

Experiment  <p.  Apply  Experiments  6  and  7,  Lesson 
3,  to  some  of  the  fibrin,  showing  it  to  be  a  protein 
substance. 

Experiment  10.  Place  a  thread  of  fibrin  in  some 
hydrogen  peroxide  (H302).  A  rapid  evolution  of  oxy- 
gen gas  is  the  result.  The  oxygen  may  be  tested  for 
by  its  relighting  a  spark  on  the  end  of  a  match. 

Experiment  11.     Mix  a  drop  of  defibrinated  blood  of 


22  LESSON    V. 

a  guinea  pig  on  a  slide  with  a  drop  of  water,  put  on  a 
cover-glass,  and  in  a  few  minutes  the  corpuscles  are 
rendered  colorless,  and  then  the  oxyhemoglobin  crys- 
tallizes out  as  rhombic  tetrahedra  (four-sided  pyramids). 
The  form  of  the  crystals  varies  with  the  blood  from 
different  animals. 

Experimetit  12.  Treat  a  little  of  the  haemoglobin 
given  with  a  drop  or  two  of  nitric  acid  (HNOs)  on  a 
watch  glass,  and  apply  gentle  heat  to  dryness.  When 
cold,  test  the  presence  of  iron  in  the  residue  by  means  of 
a  drop  of  ammonium  sulphocyanide(NH4CNS),  which 
gives  a  blood-red  coloration  in  the  presence  of  iron. 

Experiment  ij.  Observe  the  estimation  of  haemo- 
globin in  a  specimen  of  blood  by  means  of  a  haemo- 
globinometer. 

Experiment  i/f..  Make  a  dilute  blood  solution  in  a 
test  tube,  and  place  it  in  front  of  the  slit  of  the  spec- 
troscope, allowing  the  light  to  pass  through  the  solu- 
tion. 

An  absorption  spectra  will  be  seen  with  two  (2) 
absorption  bands  between  the  Fraunhofer  lines  D  and 
E,  namely,  in  the  yellow  and  green  parts  of  the 
spectrum. 

This  absorption  is  produced  by  the  oxyhaemoglobin. 

Experiment  15.  To  another  portion  of  dilute  blood 
add  some  Stoke's  reducing  solution  (an  ammoniacal 
solution  of  iron  tartrate).  Observe  the  change  of  color 
of  the  solution,  and  place  it  in  front  of  the  spectroscope 
as  before. 

An  absorption  spectra  will  be  seen  with  only  one  (1) 
broad  absorption  band  between  the  Fraunhofer  lines 
D  and  E. 


BLOOD  AND  BILE.  23 

This  absorption  is  produced  by  haemoglobin  or  re- 
duced oxyhemoglobin. 

Experiment  16.  Place  a  drop  of  blood  on  a  slide, 
and  add  a  grain  of  common  salt,  and  cover  with  a 
cover-glass.  Now  add  some  glacial  acetic  acid  under 
the  cover-glass,  and  warm  gently  until  bubbles  rise. 
When  viewed  with  the  microscope,  dark  brown,  long 
rhombic  crystals  of  hsemin  are  seen.  If  no  crystals 
appear  after  the  first  heating,  warm  again,  and,  if 
necessary,  add  some  more  acetic  acid.  These  crystals 
are  sometimes  called  Teichmann's  crystals,  and  are 
characteristic  of  blood-coloring  matters. 

Experiment  ij.  Add  some  tincture  of  guaiacum  to 
some  dilute  blood  solution  in  a  test  tube,  and  then  a 
drop  or  two  of  hydrogen  peroxide  (H2Os).  A  bluish- 
green,  and  then  a  blue  coloration  is  obtained.  A 
frothing  of  the  liquid  is  also  observed,  due  to  the  evolu- 
tion of  oxygen  from  the  hydrogen  peroxide. 

Experiment  18.  Apply  the  same  test  to  a  blood 
stain  on  a  piece  of  cloth.  The  same  coloration  is  ob- 
tained as  above. 

BILE. 

Experi?nent  ig.  Pettenkofer's  test  for  bile  acids. 
Treat  a  few  drops  of  bile  in  a  porcelain  dish  with 
a  few  drops  concentrated  sulphuric  acid  (H2S04)  and 
warm  gently.  Then  add  a  few  drops  of  a  ten-per-cent. 
solution  of  cane  sugar,  continually  stirring  with  a  glass 
rod.  A  beautiful  red  liquid,  which  gradually  changes 
to  bluish  violet,  is  obtained.  This  shows  the  presence 
of  bile  acids  (glycocholic  and  tauracholic  acids). 


24  LESSON    V. 

Experiment  20.     Gmelin  s  test  for  bile  pigments. 

Place  some  yellow  nitric  acid  (containing  nitrous  acid) 
in  a  test  tube  and  allow  some  bile  to  flow  down  gently 
on  its  surface.  A  series  of  colored  layers  are  obtained 
at  the  juncture  of  the  two  liquids  in  the  following 
order  from  above  downwards:  green,  blue,  violet,  red, 
and  reddish-yellow.  The  green  ring  must  never  be 
absent,  otherwise  the  reaction  is  not  characteristic  of 
bile  pigments. 

Experiment  21.     H upper? s  test  for  bile  pigments. 

Add  some  calcium  chloride  (CaCl2)  solution  to  some 
bile  solution  in  a  test  tube  and  precipitate  with  am- 
monium hydrate  (NH4OH).  Filter  off  the  precipitate, 
wash  with  a  little  water,  transfer  while  moist  to  a  test 
tube,  and  treat  with  alcohol  which  has  been  acidified 
with  sulphuric  acid.  Now  boil  for  some  time,  when 
the  solution  becomes  emerald-green  or  bluish-green  in 
color. 

Experiment  22.     Smith's  test  for  bile  pigments. 

Place  some  bile  solution  in  a  test  tube  and  allow  some 
tincture  of  iodine  to  flow  on  its  surface.  A  green  ring 
is  seen  at  the  juncture  of  the  two  liquids. 

Experiment  23.     Cholesterine. 

Treat  a  crystal  on  a  watch  glass  with  a  mixture  of 
5  parts  sulphuric  acid  and  1  part  water,  when  colored 
rings  are  obtained,  first  a  bright  carmine-red  and  then 
violet. 


Lesson  VI. 

MILK. 

Consists  of  an  emulsion  of  very  finely  divided  fat 
globules  suspended  in  a  solution  of  albuminous  bodies, 
milk  sugar,  and  salts  in  water. 

Experiment  I.  Test  the  reaction  of  the  milk  given 
by  means  of  litmus  paper.  It  may  be  amphoteric,  that 
is,  it  may  turn  blue  litmus  paper  red,  and  at  the  same 
time  turn  red  paper  blue,  showing  it  to  have  both  an 
acid  and  an  alkaline  reaction  to  litmus. 

Experiment  2.  Take  the  specific  gravity  of  the  milk 
by  means  of  the  hydrometer  at  a  temperature  of  150  C. 

Pure  milk  should  have  a  specific  gravity  of  1028  to 
1034. 

Experiment  j.  Mix  a  little  water  with  some  milk 
(about  £  its  volume)  and  take  the  specific  gravity  at 
the  same  temperature  as  before.  The  specific  gravity 
is  diminished  depending  upon  the  amount  of  water 
added. 

Experiment  4.  Boil  some  of  the  milk  in  a  test  tube. 
No  coagulation  or  change  is  observed,  with  the  excep- 
tion of  the  formation  of  a  skin  on  the  surface. 

Experiment  5. — Slightly  acidify  a  little  of  the  milk 
with  acetic  acid  and  then  warm.  An  immediate  coagu- 
lation or  curdling  of  the  casein  is  produced. 

25 


26  LESSON    VI. 

Experiment  6.  Warm  some  milk  up  to  400  C.  and 
add  some  rennet  powder  or  rennin.  The  casein  coagu- 
lates immediately,  forming  a  clot,  and  a  clear  liquid 
called  the  "whey"  separates.  Filter  the  liquid  off 
and  retain  it  for  further  experiments. 

This  process  is  made  use  of  in  the  making  of  cheese. 

Experiment  7.  Heat  a  little  of  the  clot  on  foil.  It 
burns  with  an  odor  of  burnt  hair,  showing  it  to  be 
nitrogenized. 

Experiment  8.  Apply  the  Biuret  test  to  a  portion  of 
the  clot.  A  violet  reaction  is  obtained  showing  it  to 
be  a  protein  body. 

Experiment  p.  Shake  another  portion  of  the  clot  in 
a  test  tube  with  some  ether.  Decant  the  ether  into  a 
watch  glass,  and  let  it  evaporate.  A  residue  of  fats 
will  be  found  in  the  watch  glass.  This  shows  that  the 
casein  in  coagulating  carries  with  it  most  of  the  fats  of 
the  milk. 

Experiment  10.  Add  some  copper  sulphate  (CuS04) 
solution  to  some  milk  in  a  test  tube,  and  then  one  drop 
of  caustic  soda  (NaOH)  solution.  A  precipitate  of  the 
casein,  carrying  most  of  the  fats  with  it,  is  produced. 
This  is  the  basis  of  Ritthausen's  method  for  the  esti- 
mation of  casein  in  milk. 

Experiment  11.  Keat  some  of  the  "  whey  "  obtained 
in  Experiment  6  with  some  Fehling's  solution. 

A  copious  reduction  of  copper  sub-oxide  shows  the 
presence  of  milk  sugar  (lactose).  The  "  whey  "  should 
have  a  sweetish  taste. 

Experiment  12.  Witness  the  estimation  of  fat  in  a 
sample  of  milk  by  means  of  the  Lactrocrit,  and  also  by 
Soxhlet's  extraction  apparatus. 


MILK.  27 

Experinunt  ij.  Place  some  milk  in  a  narrow  test 
tube,  and  place  it  in  the  centrifugal  machine  and  rotate 
for  a  few  minutes.  The  cream,  or  fat,  is  made  to  rise 
to  the  surface,  and  should  occupy  from  ten  to  fifteen 
per  cent,  of  the  column  of  the  milk.  The  same  result 
may  be  obtained  by  allowing  the  milk  to  stand  in  the 
test  tube  for  twelve  to  twenty-four  hours. 

Experiment  14.  Place  a  drop  of  the  milk  on  a  slide 
and  cover  with  a  cover-glass.  View  this  under  the 
microscope  and  observe  the  number  and  size  of  the 
fat  globules. 


Lesson  VII. 
URINE. 

i.  Collection  and  measurement  of  the  twenty-four 
hours'  urine.  Start  at  a  given  time  with  an  empty 
bladder,  and  then  collect  all  the  urine  excreted  in  the 
following  twenty-four  hours. 

Average  normal  amount  for  man  about  1500  c.  c. 
Women  a  little  less.  The  quantity  of  drink  greatly  in- 
fluences the  quantity  of  urine  voided. 

2.  Color,  normally  amber-yellow.  (Refer  to  Vogel's 
color  chart.) 

3.  Transparency,  Clear,  with  the  exception  of  a 
slight  cloud  of  mucus,  which  suspends  itself  midway  in 
the  liquid  on  allowing  it  to  stand. 

4.  Consistency.     Not  ropy. 

5.  Odor.  Urinous,  but  sometimes  certain  foods  and 
medicines  give  a  characteristic  odor. 

6.  Reaction.  Normally  slightly  acid  for  the  twenty- 
four  hours.  Portions  voided  at  different  times  of  the 
twenty-four  hours  and  after  certain  food  have  different 
reactions.  The  reaction  is  determined  by  placing  some 
of  the  urine  on  litmus  paper.  If  it  be  acid,  the  blue 
paper  will  turn  red.  If  alkaline,  the  red  paper  will  turn 
blue. 

The  alkalinity  may  be  due  to  the  fixed  alkalies,  such 

28 


URINE.  29 

as  sodium  or  potassium  carbonates  or  phosphates,  or 
to  volatile  alkali,  such  as  ammonium  carbonate. 

If  the  alkalinity  is  due  to  the  volatile  alkali,  on  sus- 
pending the  litmus  paper,  which  has  turned  blue,  to 
the  air,  the  original  red  will  come  back  again.  If  the 
alkalinity  be  due  to  the  fixed  alkalies,  the  blue  color 
will  be  permanent. 

Experiment.  Test  the  reaction  of  the  sample  of 
urine  given.  If  alkaline,  determine  if  it  is  due  to  fixed 
or  volatile  alkalies,  as  above  described. 

Experiment.  If  the  alkalinity  is  due  to  volatile  al- 
kali, it  will  have  an  odor  of  ammonia  and  will  give 
white  fumes  of  ammonium  chloride  (NH4C1)  if  a  glass 
rod,  moistened  with  hydrochloric  acid,  is  held  over  the 
urine. 

7.  Specific  Gravity.  Normally  about  1020  at6o°  F., 
or  1 5. 50  C.  The  specific  gravity  varies  generally  with 
the  quantity  of  urine  voided,  but  not  always. 

It  is  generally  determined  by  means  of  a  urinometer 
or  specific  gravity  bottle.  Always  determine  the  spe- 
cific gravity  at  60  °  F.  or  15. 50  C,  taking  care  not  to 
have  any  air  bubbles  attach  themselves  to  the  instru- 
ment, and  to  have  the  vessel,  holding  the  urine,  large 
enough  so  that  the  instrument  floats  readily.  The 
vessel  should  not  be  held  by  the  hand,  but  should  be 
allowed  to  stand  in  a  vertical  position.  It  should  be 
filled  to  the  top  with  urine. 

Read  off  on  the  spindle  of  the  instrument  where  the 
meniscus  of  the  liquid  strikes  the  graduation. 

Experiment.  Determine  the  specific  gravity  of  the 
sample  of  urine  given. 

Experiment.     Dilute  a  given  volume  of  this  urine 


30  LESSON    VII. 

with  an  equal  volume  of  water,  and  mix  thoroughly. 
Take  the  specific  gravity  of  the  mixture.  It  will  be 
found  to  be  one  half  of  the  original  urine.  If  the  spe- 
cific gravity  of  the  mixture  of  one  volume  of  urine  and 
two  volumes  of  water  is  taken,  it  will  be  found  to  be 
one  third  of  the  original  urine. 

In  cases  where  too  little  urine  is  voided  to  take  the 
specific  gravity,  the  urine  can  be  diluted  with  a  known 
volume  of  water,  and  then  the  specific  gravity  taken, 
and  from  this  calculate  the  specific  gravity  of  the  origi- 
nal urine. 

8.  Total  Solids.  The  approximate  amount  of  solids 
may  be  calculated  from  the  specific  gravity  by  multi- 
plying the  last  two  figures  of  the  specific  gravity  when 
below  1018  by  2  or  when  above  by  2.33.  The  result  is 
parts  of  solid  in  1000  parts  of  the  urine. 

Experiment.  Make  the  calculation  for  the  urine 
given,  supposing  in  one  case  the  total  quantity  of  urine 
voided  in  the  twenty-four  hours  was  1450  c.  c.  and  in 
the  other  case  850  c.  c. 

Experiment.  Heat  some  of  the  evaporated  urine 
given  on  the  foil.  It  blackens  and  burns  with  an  odor 
of  burnt  hair,  due  to  the  nitrogenous  organic  matter. 
On  continuing  the  heat  all  the  organic  matter  burns 
away,  leaving  a  white  residue  which  consists  of  the 
inorganic  salts. 

NORMAL   PROPORTIONS. 

Total     solids...  .60  grammes  in  the  24  hours. 

Organic     " 35         "  "     "     " 

Inorganic"     ...,25         "  "     "     " 


URINE.  31 

Organic  Solids.  Inorganic  Solids. 

Urea 30    grms.      Sodium  chloride..  15  grms. 

Uric  acid 0.7    "  Sulphuric  acid.  ..  .2.5  " 

Creatinin 1.0    "  Phosphoric  acid.  .2.5  " 

Hippuric  acid...  .0.7     "  Potash 3.3  " 

Remaining  organ-  Ammonia 0.7  " 

ic  bodies 2.6    "  Magnesia 0.5  " 

Lime 0.3  " 

Remaining   inor- 
ganic bodies.  .  .0.2  " 

UREA,    CH4N20. 

Colorless,  crystalline  needles  or  prisms. 
-    Very  soluble  in   water  or  alcohol.     It   is  soluble  in 
its  own  weight  of  water  and  five  times  its  weight  of 
alcohol. 

Experiment.  Try  the  solubility  of  the  urea  given  in 
water.     Retain  this  solution  for  further  use. 

Experiment.  Try  the  solubility  of  the  urea  in  alco- 
hol. Place  a  drop  of  this  alcoholic  solution  on  a  clean 
microscope  slide  and  observe  the  crystallization. 

Urea  is  a  basic  compound  forming  salts  with  acids, 
such  as  urea  nitrate  CH4N20,HN03  or  urea  oxalate, 
CH4N20,  C2H204.  which  are  crystalline. 

Experiment.  Place  a  little  of  the  watery  solution  of 
urea  obtained  above  in  a  watch  glass  and  add  a  few 
drops  of  nitric  acid  thereto.  Hexagonal  crystalline 
plates  of  urea  nitrate  are  formed  in  the  liquid. 

This  compound  may  be  employed  in  the  detection  of 
small  amounts  of  urea  in  liquids. 

Experiment.     Treat    some  of  the  evaporated  urine 


32  LESSON    VII. 

given  with  a  few  drops  nitric  acid  and  observe  the  for- 
mation of  the  crystals  of  urea  nitrate. 

Urea  readily  changes  into  ammonium  carbonate  by 
the  action  of  chemical  processes  or  micro-organisms. 
(This  latter  is  called  alkaline  fermentation  of  the  urine.) 
CH4NsO  +  2H  20=(NH4)2C03. 

This  change  may  take  place  in  the  body  and  the 
urine  voided  under  such  circumstances  will  be  alkaline 
due  to  volatile  alkali,  will  probably  have  an  odor  of 
ammonia  and  give  white  fumes  of  ammonium  chloride 
with  a  drop  of  hydrochloric  acid,  if  held  over  it  on  a 
glass  rod. 

Experiment.  Heat  a  little  urea  in  a  dry  test  tube. 
It  melts,  decomposes,  and  gives  off  ammonia  (NH3), 
and  leaves  finally  a  white  mass,  which  if  dissolved  in 
water  and  treated  with  a  drop  of  copper  sulphate 
(CuS04)  and  an  excess  of  caustic  soda,  gives  a  beauti- 
ful reddish-violet  liquid.     (Biuret  reaction.) 

Urea  is  decomposed  by  an  alkaline  solution  of  sodium 
hypochlorite,  or,  better,  hypobromite  into  nitrogen,  car- 
bon dioxide,  and  water,  according  to  the  following 
equation  : 

CH4N20+3NaBrO  =  N2+C03+2H30+3NaBr. 

Experiment.  Place  a  little  urea  solution  in  a  test 
tube  and  add  some  sodium  hypobromite.  A  violent 
decomposition  takes  place  with  the  rapid  evolution  of 
gas.  Let  the  gas  bubbles  subside  and  apply  a  lighted 
match  to  the  gas  in  the  test  tube.  The  flame  is  extin- 
guished, due  to  the  nitrogen,  which  is  a  non-supporter 
of  combustion.  The  carbon  dioxide  (C02)  evolved  is 
absorbed  by  the  alkaline  solution  of  sodium  hypo- 
bromite. 


URINE.  33 

Experiment.  Quantitative  Estimation.  Fill  the  nre- 
ometer  with  sodium  hypobromite  (prepared  as  described 
below)  by  holding  it  in  a  vertical  position  and  pouring 
the  solution  into  the  bulb,  and  when  it  is  rather  more 
than  one  half  full,  incline  the  instrument  horizontally 
until  the  entire  tube  is  filled  and  a  little  left  in  the  bulb, 
say  about  one  third,  when  in  the  vertical  position. 
Now  fill  the  nipple  pipette  up  to  the  I  c.  c.  mark 
with  urine  and  slozvly  inject  this  amount  of  urine  up 
under  the  tube  and  not  in  the  bulb  of  the  instrument. 
Allow  frothing  to  cease  and  let  it  stand  a  moment  or 
two,  so  that  the  carbon  dioxide  may  be  absorbed. 
Read  off  on  the  graduation  the  weight  of  urea  corres- 
ponding to  the  volume  of  nitrogen  collected.  The  in- 
struments are  graduated  to  read  fractions  of  a  gramme 
of  urea  per  cubic  centimeter  of  urine. 

The  solution  of  sodium  hybromite  is  prepared  as 
follows : 

Dissolve  ioo  grammes  caustic  soda  in  250  c.  c.  water, 
and  adding  25  c.  c.  bromine  to  this  solution  when  cold. 
For  use  in  the  ureometer  this  solution  should  be  diluted 
with  an  equal  volume  of  water. 

As  this  solution  does  not  keep  for  any  length  of 
time,  a  solution  has  been  suggested  by  Dr.  Rice  as 
follows : 

1.  Dissolve  125  grammes  bromine,  125  grammes 
sodium  bromide  in  1000  c.  c.  water. 

2.  Prepare  a  solution  of  caustic  soda  of  a  specific 
gravity  of  1.250. 

For  use  in  Doremus's  ureometer  mix  5  c.  c.  of  each 
of  the  two  above  solutions  and  dilute  with  15  c.  c. 
water. 


34 


LESSON    VII. 


URIC  ACID  C5H4N403  or  H2C5H2N403. 

Is  a  bibasic  acid,  thus  forming  two  series  of  salts.  Ex- 
ample : 

Acid  sodium  urate,  NaHC5H2N403. 
Neutral  "  "       Na2C5H2N403. 

Soluble  with  difficulty  in  cold  water  (i  part  in  15,000 
parts)  and  more  soluble  in  boiling  water  (1  part  in  1900 
parts).  The  acid  salts  are  less  soluble  than  the  neutral 
salts.  The  alkali  salts  are  more  soluble  than  the 
earthy,  such  as  calcium  or  magnesium.  Ammonium 
urate  is  very  insoluble.  The  uric  acid  exists  in  the 
urine  as  urates  and  never  free.  Sodium  phosphate  is 
considered  as  the  solvent  for  the  uric  acid  in  the  urine. 

Uric  acid  may  occur  in  nearly  every  crystalline  form, 
but  generally  as  small  rhombical  prisms  or  plates,  dumb- 
bell shaped  crystals,  whetstone  shaped,  etc.  When 
deposited  from  the  urine  they  are  always  more  or  less 
colored. 

Experiment.  Place  a  little  uric  acid  in  a  test  tube 
and  add  some  water.  Note  insolubility.  Heat  to  boil- 
ing and  notice  the  apparent  insolubility. 

Experiment.  Add  a  few  drops  of  sodium  carbonate 
to  the  above  solution.  The  uric  acid  dissolves  readily 
with  the  formation  of  sodium  urate. 

Experiment.  Add  a  few  drops  of  hydrochloric  acid 
to  a  portion  of  the  above  solution  of  sodium  urate.  A 
white  precipitate  of  uric  acid  is  produced.  The  HC1 
added  combines  with  the  sodium,  setting  the  insoluble 
uric  acid  free. 

Experiment.  Saturate  some  of  the  solution  of  uric 
acid  in  sodium  carbonate  with  ammonium  chloride.     A 


URINE.  35 

white  gelatinous  precipitate  of  insoluble  ammonium 
urate  is  formed.  (This  is  the  basis  of  Hopkin's  method 
of  estimating  uric  acid  in  urine.) 

Experiment.  Murexid  test.  Place  a  few  grains  of 
the  uric  acid  on  a  watch  glass,  moisten  with  a  drop  of 
nitric  acid,  and  gently  heat  to  dryness  over  flame.  A 
yellowish-red  residue  is  obtained.  When  cold  add  a 
drop  of  ammonium  hydrate.  A  purple  coloration  is 
the  result,  which  becomes  darker  on  the  addition  of 
caustic  soda.  The  purple  color  is  ammonium  pur- 
purate. 

Experiment.  Schiff's  test.  Dissolve  a  little  of  the 
uric  acid  in  water,  to  which  some  sodium  carbonate 
(Na2C03)  has  been  added.  Add  a  drop  of  silver  ni- 
trate (AgN03)  to  a  portion  of  the  solution.  A  black 
reduction  of  silver  oxide  is  obtained.  The  test  may 
also  be  performed  by  applying  a  drop  of  the  solution 
of  uric  acid  to  a  piece  of  paper  previously  moistened 
with  silver  nitrate  (AgN03).  A  black  stain  appears 
on  the  paper. 

Experiment.  Place  a  little  Fehling's  solution  in  a 
test  tube,  and  add  a  little  of  the  uric  acid  solution.  A 
white  precipitate  of  copper  urate  is  produced,  which  if 
heated  is  reduced  to  red  copper  sub-oxide.  This  should 
be  considered  in  testing  for  sugar  (dextrose)  in  the 
urine.  Alkaline  solutions  of  bismuth  are  not  reduced 
by  uric  acid. 

Experiment.  Place  some  uric  acid  on  foil  and  apply 
heat.  It  blackens,  gives  off  an  odor  of  burnt  hair,  and 
is  entirely  consumed,  showing  it  to  be  entirely  of  an 
organic  nitrogenous  nature. 

Experiment.     Heat  some  ammonium  urate  (  (NH4)3 


36  -  LESSON    VII. 

C5H3N403)  on  foil.  It  blackens,  gives  off  an  odor  of 
burnt  hair,  and  entirely  disappears. 

Experiment.  Heat  some  potassium  urate  (K3C5H0 
N4Os)  on  the  foil.  It  blackens,  gives  off  an  odor  of 
burnt  hair,  and  leaves  a  residue  of  potassium  carbonate 
(K3C03),  which,  if  placed  on  moistened  red  litmus 
paper,  turns  it  blue  because  of  its  alkalinity. 

Quantitative  Estimation.  Acidify  200  c.c.  of  the 
urine  previously  filtered  and  free  from  albumin,  with 
10  c.c.  hydrochloric  acid  in  a  beaker,  stir  and  allow  this 
mixture  to  stand  in  a  cool  place  for  twenty-four  to 
forty-eight  hours.  Filter  off  the  precipitated  uric  acid 
through  a  previously  weighed  filter,  taking  care  that 
all  the  particles  of  uric  acid  are  removed  from  the  sides 
of  the  glass  vessel  and  transferred  to  the  filter.  Wash 
the  precipitate  with  water  until  it  is  free  from  chlorine, 
as  shown  by  adding  silver  nitrate  to  a  portion  of  the 
filtrate.     Now  dry  and  weigh. 

As  some  uric  acid  is  dissolved  by  the  wash  water, 
the  filtrate  and  wash  water  should  be  measured,  and 
for  each  10  c.c.  of  filtrate  and  wash  water  0.00048  grms. 
uric  acid  must  be  added  to  the  amount  found  by 
weighing. 

Hopkins  Method.  Treat  100  c.c.  of  the  urine  in  a 
beaker  with  30  grms.  powdered  ammonium  chloride 
(NH4C1),  and  allow  to  stand  in  a  cool  place  for  twenty- 
four  hours.  Filter  the  precipitated  ammonium  urate 
through  a  small  filter,  and  wash  with  a  cold  saturated 
solution  of  ammonium  chloride.  Puncture  the  filter 
and  wash  the  precipitate  into  a  beaker  with  a  small 
amount  of  boiling  water,  and  treat  with  5  c.c.  dilute 
hydrochloric  acid  and  heat  on  the  water  bath.     When 


URINE.  37 

cold  filter  the  uric  acid  through  a  weighed  filter  and 
proceed  as  above  described. 

HIPPURIC  ACID,  C9H9N03. 

Crystallizes  in  semi-transparent,  milk-white,  long, 
four-sided  rhombic  prisms  or  columns,  or  in  needles  on 
rapid  crystallization. 

Hippuric  acid  dissolves  in  600  parts  cold  water  and 
more  easily  in  hot  water.  It  is  soluble  in  alcohol.  It 
is  a  monobasic  acid  forming  hippurates  with  bases, 
which  are  soluble  in  water  and  alcohol. 

Experiment.  Boil  a  watery  solution  of  hippuric 
acid  with  a  drop  or  two  of  hydrochloric  acid.  It  is 
split  into  benzoic  acid  (C6H5COOH)  and  glycocoll 
(C2H5N02).  This  change  may  also  be  brought  about 
by  certain  micro-organisms. 

Experiment.  Add  some  ferric  chloride  (Fe2Cl,.)  to 
some  of  the  above  solution.  A  bluish-red  coloration 
shows  the  presence  of  benzoic  acid. 

Experiment.  Heat  some  of  the  hippuric  acid  (not 
solution)  with  nitric  acid  (HN03)  in  a  test  tube.  An 
odor  of  oil  of  bitter  almonds  (nitro-benzole)  is  de- 
veloped. 

Other  organic  bodies  occurring  in  the  urine : 

Creatinin C4H,N30 

Oxalic  acid C2H204 

Allantoin C4H6N403 

Xanthin C5H4N402 

Hypoxanthin C5H4N40 

Guanin C5H5NgO 

Adenin C5H5N5 


Lesson  VIII. 


URINE. 


Occur  chiefly  as  sodium  chloride  (NaCl)  with  small 
amounts  of  potassium  chloride.  They  are  estimated 
as  sodium  chloride.  Sodium  chloride  is  very  soluble 
in  cold  and  hot  water,  hence  it  never  occurs  in  urinary 
sediments  or  calculi. 

Experiment.  Add  some  silver  nitrate  to  some  dilute 
hydrochloric  acid  in  a  test  tube.  A  white  precipitate 
of  silver  chloride  (AgCl)  is  produced.  Divide  into  two 
portions.  Add  ammonium  hydrate  (NH4OH)  to  one 
portion  and  notice  that  the  precipitate  dissolves.  Add 
nitric  acid  (HN03)  to  the  other  portion.  It  does  not 
dissolve.  * 

Experiment.  Place  some  sodium  phosphate  (Na2- 
HP04)  in  a  test  tube  and  add  some  silver  nitrate 
(AgN03).  A  yellow  precipitate  of  silver  phosphate  is 
formed.  Add  nitric  acid  (HNOs)  to  this  and  observe 
that  the  precipitate  is  dissolved. 

Experiment.  To  detect  chlorides  in  a  sample  of 
urine,  first  add  a  few  drops  nitric  acid  and  then  silver 
nitrate,  so  as  to  prevent  the  phosphates  from  being 
precipitated  by  the  silver  nitrate,  and  in  the  presence 
of  chlorides  a  white  precipitate  is  obtained. 

Quantitative  Estimation.     Place  20  c.c.  of  the  urine 

38 


URINE.  39 

in  a  clean  porcelain  dish  and  add  thereto,  drop  by 
drop,  a  standard  solution  of  silver  nitrate  (see  below) 
from  a  burette,  constantly  stirring  with  a  glass  rod. 
From  time  to  time  place  a  drop  of  the  contents  of  the 
dish  on  a  piece  of  paper  previously  moistened  with  a 
solution  of  potassium  chromate.  When  a  faint  red 
stain  is  obtained  on  this  paper  stop  adding  the  silver 
nitrate  solution.  This  indicates  that  enough  silver 
nitrate  solution  has  been  added  to  combine  with  all 
the  chlorine  in  the  20  c.  c.  of  urine  in  the  dish.  Read 
off  the  number  of  cubic  centimetres  of  silver  nitrate 
solution  used.  Each  c.  c.  of  the  standard  solution  of 
silver  nitrate  is  equal  to  0.01  grammes  sodium  chloride 
(NaCl).  From  the  weight  of  NaCl  in  the  20  c.  c.  of 
urine  calculate  the  total  amount  in  the  twenty-four 
hours. 

The  standard  solution  of  silver  nitrate  is  prepared  by 
dissolving  29.075  grammes  fused  silver  nitrate  in  1000 
c.  c.  of  distilled  water.     Each  c.  c.  =  0.01  grms.  NaCl. 

SULPHURIC  ACID,  S03. 

The  sulphuric  acid  of  the  urine  occurs  either  in  com- 
bination with  bases  as  potassium  (K3S04)  and  sodium 
sulphate  (Na3S04)  or  as  ethereal  sulphates,  such  as 
potassium  phenol  sulphate  (C6H5.O.S02.OK),  or 
potassium  indoxyl  sulphate  (C8H6N.O.S03.OK). 

Chiefly  formed  from  the  sulphur  of  the  protein 
bodies  by  oxidation  and  a  small  portion  from  the 
sulphates  of  the  food. 

Experiment.  Place  some  magnesium  sulphate  (Mg- 
S04j   in  a  test   tube  and  add  some  barium   chloride 


40  LESSON    VIII. 

(BaCl3)  thereto.  A  white  precipitate  of  barium  sul- 
phate (BaS04)  is  produced. 

Try  the  solubility  of  this  precipitate  of  barium 
sulphate  in  nitric  acid  and  in  caustic  soda.  It  will  be 
found  insoluble  in  both. 

Experiment.  Acidify  some  urine  with  acetic  acid 
and  add  some  barium  chloride.  A  white  precipitate 
of  barium  sulphate  is  produced.  Filter  this  off  and  re- 
tain the  filtrate. 

This  precipitate  represents  the  sulphuric  acid  exist- 
ing as  potassium  and  sodium  sulphates. 

Experiment.  Add  some  hydrochloric  acid  to  the 
above  filtrate  and  boil  for  a  few  minutes.  This  de- 
composes the  ethereal  sulphates  setting  more  sul- 
phuric acid  free,  and  this  is  then  precipitated  as  white 
barium  sulphate  by  the  barium  chloride  in  the  filtrate. 

Quantitative  Estimation,  ioo  c.  c.  of  the  filtered 
urine  are  acidified  with  5  c.  c.  concentrated  hydrochloric 
acid  and  boiled  for  fifteen  minutes.  While  boiling  add 
2  c.  c.  of  a  strong  solution  of  barium  chloride  and  warm 
for  a  little  while  until  the  barium  sulphate  has  com- 
pletely settled.  Filter  through  a  small  filter  and  wash 
with  hot  water  and  then  with  alcohol  (to  remove  any 
resinous  bodies).  Dry  the  filter,  incinerate  and  weigh 
according  to  prescribed  methods.  From  the  weight  of 
barium  sulphate  calculate  the  amount  of  sulphuric  acid 
in  the  urine  used. 

PHOSPHORIC   ACID,    P305. 

It  occurs  in  urines  combined  with  sodium,  potassium, 
ammonium,  calcium,  and  magnesium,  and  as  phosphoric 
acid  is  tribasic,  three  series  of  salts  occur,  namely  acid, 


URINE.  4 1 

neutral,  and  basic  phosphates.  The  phosphates  of 
potassium,  sodium,  and  ammonium  are  called  alkali 
phosphates,  and  are  very  soluble  in  the  urine  ;  while 
the  phosphates  of  calcium  and  magnesium  are  called 
earthy  phosphates  and  are  difficultly  soluble  in  acid 
urine,  but  insoluble  in  alkaline  urines. 

The  chief  quantity  of  phosphoric  acid  is  derived 
from  the  phosphates  of  the  food,  but  a  small  amount 
is  derived  from  the  oxidation  of  organic  bodies  con- 
taining phosphorous  such  as  nuclein,  lecithin,  protagon. 

Experiment.  Place  some  sodium  phosphate  (Na2- 
HP04)  in  a  test  tube  with  water  and  add  some  magne- 
sium sulphate  (MgS04)  solution,  then  some  ammonium 
chloride  (NH4C1)  and  lastly  some  ammonium  hydrate 
(NH4OH).  A  copious  white  precipitate  of  magnesium- 
ammonium  phosphate  (MgNH4P04,6H20,  triple  phos- 
phate) is  formed.  This  is  a  good  test  for  phosphoric 
acid  or  phosphates. 

Experiment.  Add  some  caustic  soda  (NaOH)  to 
some  urine  in  a  test  tube  and  warm  gently.  A  flocu- 
lent  precipitate  of  earthy  phosphates  is  produced. 

Filter  this  precipitate  off  and  make  the  filtrate  acid 
with  acetic  acid,  add  magnesium  sulphate,  ammonium 
chloride,  and  then  ammonium  hydrate.  A  white  pre- 
cipitate of  magnesium-ammonium  phosphate  is  formed, 
derived  from  the  alkali  phosphates  in  the  urine. 

Experiment.  Add  some  ammonium  hydrate  to  some 
urine  in  a  test  tube.  A  white  precipitate  of  calcium 
phosphate  (Ca3(P04)2)  and  crystalline  magnesium- 
ammonium  phosphate  (triple  phosphate)  is  formed. 
This  forms  whenever  urine  becomes  ammoniacal,  by 
decomposition  of  urea,  on  standing. 


42  LESSON    VIII. 

Experiment.  Add  some  uranium  acetate  solution  to 
some  sodium  phosphate  in  a  test  tube.  A  white  pre- 
cipitate of  uranium  phosphate  is  obtained.  This  is  one 
of  the  best  tests  for  phosphoric  acid  or  phosphates. 

Quantitative  Estimation.  Place  50  c.  c.  of  the  filtered 
urine  in  a  clean  porcelain  dish  and  add  a  few  drops 
acetic  acid  or  better  a  solution  of  sodium  acetate. 
Warm  over  water  the  bath  and  add  a  standard  solution 
of  uranium  acetate  from  a  burette  drop  by  drop,  stirring 
all  the  while,  until  a  drop  taken  out  and  placed  on  a 
piece  of  paper  moistened  with  potassium  ferrocyanide 
(K4Fe(CN)6)  gives  a  faint  chocolate  brown  stain. 
Place  it  on  the  water  bath  again  and  warm  and  apply 
a  drop  to  the  potassium  ferrocyanide  paper.  If  the 
stain  is  permanent  stop  adding  the  uranium  acetate 
solution,  otherwise  go  on  until  a  permanent  stain  is  ob- 
tained. Each  c.  c.  of  the  standard  solution  of  uranium 
acetate  corresponds  to  0.005  grammes  phosphoric  acid 
(Ps06).  From  the  above  result  calculate  the  total 
amount  of  phosphoric  acid  in  the  twenty-four  hours 
urine. 

Experiment.  To  determine  the  P205  in  combination 
as  alkali  or  earthy  phosphates  separately,  first  precipi- 
tate the  earthy  phosphates  from  100  c.  c.  of  the  urine 
by  the  addition  of  a  very  small  amount  of  ammonium 
hydrate.  Filter  this  precipitate  off  and  collect  50  c.  c. 
of  the  filtrate.  Acidify  this  with  acetic  acid  and  deter- 
mine the  quantity  of  P2Og  therein  by  means  of  the 
standard  solution  of  uranium  acetate  as  above  directed. 
This  gives  the  amount  of  P2Og  combined  with  sodium 
and  potassium  or  alkali  phosphates.  On  subtracting 
this  result  from  the  total  amount  of  P3Og  in  the  urine 


URINE.  43 

we  get  the  amount  of  P2Og  existing  in  combination 
with  calcium  and  magnesium,  or  earthy  phosphates. 

ABNORMAL   CONSTITUENTS.— PROTEIN   SUBSTANCES. 
Abnormal  urine  may  contain  ser-albumin,  ser-globu- 
lin,  peptones,  mucin,  fibrin,  etc.,  but  the  most  common 
substance  is  ser-albumin. 

SER-ALBUMIN. 

In  testing  the  presence  of  albumin  in  a  sample 
of  urine  it  should  be  perfectly  clear,  and  if  not,  it 
must  first  be  clarified  by  filtration  through  one  or 
more  folds  of  filter  paper.  If  it  cannot  be  clarified  by 
simple  filtration  (due  to  the  presence  of  micro-organ- 
isms), add  some  magnesium  sulphate  (MgS04),  ammo- 
nium chloride  (NH4C1),  and  then  some  ammonium 
hydrate  (NH4OH),  forming  a  precipitate  of  triple 
phosphate,  which  retains  the  bacteria,  and  which  may 
be  removed  by  filtration.  The  filtrate  must  be  made 
acid  with  acetic  acid  before  applying  the  tests  for 
albumin. 

Before  applying  the  tests  to  a  sample  of  urine  first 
test  the  reaction  of  the  urine,  and  if  alkaline  make  it 
neutral  or  slightly  acid  with  acetic  acid. 

Experiment.  Fill  a  test  tube  two  thirds  full  with 
some  of  the  clear  urine  and  apply  heat  to  the  upper 
layer  of  the  liquid,  holding  the  test  tube  at  the  bottom. 
A  precipitation  or  coagulation  may  occur,  but  it  may 
be  due  to  earthy  phosphates  or  albumin,  or  both,  be- 
cause they  are  precipitated  by  heat  alone. 

Now  add  three  (3)  drops  of  nitric  acid  to  the  liquid. 
If  the  precipitate  dissolves  completely  on  the  addition 
of  the  acid  it  is  due  to  earthy  phosphates,  but  if  it  does 


44  LESSON    FIJI. 

not,  and  the  urine  remains  opalescent  and  cloudy,  it 
contains  albumin.  Boil  again  and  observe  if  the  pre- 
cipitate increases. 

Experiment.  Heller  s  test.  Place  about  one  half  an 
inch  of  nitric  acid  in  a  test  tube  and  allow  the  urine  to 
flow  gently  on  the  surface  of  the  acid  without  mixing 
with  it.  This  may  be  done  by  allowing  the  urine  to 
flow  from  a  filter,  holding  the  point  of  the  funnel  on 
the  side  of  the  test  tube,  inclining  it  slightly.  The 
urine  may  also  be  floated  on  the  surface  of  the  acid  by 
means  of  a  nipple  pipette,  holding  the  test  tube  in  an 
inclining  position. 

If  albumin  is  present  a  sharply-defined  opalescent 
ring  or  zone  will  be  observed  at  the  point  of  contact 
between  the  two  liquids.  In  the  presence  of  traces  of 
albumin  some  time  is  required  for  the  development  of 
the  zone.  With  a  urine  containing  an  excess  of  uric 
acid  a  zone  is  obtained,  but  it  is  higher  up  in  the  liquid 
and  not  sharply  defined  as  the  albumin  zone. 

The  ring  due  to  coloring  matters  should  not  be  con- 
founded for  the  albumin  zone. 

Experiment.  Fill  a  test  tube  one  half  full  of  clear 
urine  and  add  some  clear  potassium  ferrocyanide 
(K4Fe(CN)6)  solution  and  mix  thoroughly.  Now  add 
ten  drops  acetic  acid,  and  if  albumin  is  present  a  white 
cloudiness  or  precipitate  is  obtained.  The  obtainment 
of  a  cloudiness  with  this  test  as  above  directed  shows 
the  presence  of  albumin  and  nothing  but  albumin. 

It  is  advisable  in  all  these  tests,  in  the  presence  of 
faint  traces  of  albumin,  to  have  a  second  test  tube  con- 
taining the  same  amount  of  clear  urine,  without  any 
reagents,  and  hold  the  two  test  tubes  side  by  side  and 


URINE.  45 

look  through  the  liquid  at  a  dark  background.  By 
this  means  the  faintest  cloudiness  may  be  detected  in 
the  test  tube  to  which  the  reagents  have  been  added 
by  difference  in  appearance. 

Experiment.  Tanret's  test.  Acidify  some  of  the 
urine  in  a  test  tube  with  a  few  drops  of  acetic  acid,  and 
add  a  few  drops  of  a  solution  of  double  iodide  of  mer- 
cury and  potassium.  A  white  cloudiness  or  precipitate 
is  obtained  in  the  presence  of  albumin.  This  test  may 
also  be  applied  by  the  contact  method  ;  the  reagent  is 
placed  first  in  the  test  tube,  and  then  the  urine  allowed 
to  float  on  top.  A  white  ring  or  zone  at  the  point  of 
contact  of  the  two  liquids  is  the  result  in  the  presence 
of  albumin. 

The  reagent  is  prepared  by  adding  potassium  iodide 
to  some  mercuric  chloride  solution  in  a  test  tube  and 
just  re-dissolving  the  red  precipitate  of  mercuric  iodide 
formed  by  a  slight  excess  of  potassium  iodide  solution. 

This  test  is  very  delicate,  but  peptones,  mucin,  vege- 
table alkaloids  also  respond  to  it.  The  precipitate 
obtained  with  peptones  and  alkaloids  is  dissolved  by 
the  application  of  a  gentle  heat  and  reappears  on  cool- 
ing again. 

Experiment.  Robert's  Nitric-Magnesium  test.  Float 
the  urine  on  the  surface  of  a  solution  of  magnesium 
sulphate  containing  nitric  acid.  In  the  presence  of 
albumin  a  sharply-defined  zone  or  ring  is  seen  between 
the  two  liquids. 

The  solution  is  made  by  adding  one  ounce  of  strong 
nitric  acid  to  five  ounces  of  a  saturated  solution  of  mag- 
nesium sulpate.  This  modification  is  said  to  be  more 
delicate  than  Heller's  test. 


46  LESSON    VIII. 

Experiment.  Acidify  some  of  the  clear  urine  with 
acetic  acid  and  add  a  few  drops  of  a  picric  acid  solution. 
A  turbidity  or  cloudiness  is  seen  as  the  picric  acid  dif- 
fuses through  the  liquid. 

Peptones,  alkaloids,  such  as  quinine,  morphine,  etc., 
are  precipitated  by  this  reagent,  but  the  precipitate 
disappears  on  the  application  of  heat  and  reappears  on 
cooling  again. 

Experiment.  Make  some  of  the  urine  acid  with 
acetic  acid,  and  add  a  few  drops  of  a  solution  of  sodi- 
um tungstate.  In  the  presence  of  albumin  a  cloudi- 
ness or  precipitate  is  observed.  In  addition  to  albumin 
this  reagent  precipitates  peptones  and  mucin  but  not 
the  alkaloids.  The  precipitate  obtained  with  peptones 
is  dissolved  by  gentle  heat. 

Quantitative  Estimation.  Heat  50  c.c.  water  contain- 
ing 10  c.c.  acetic  acid  to  boiling,  and  add  gradually, 
while  boiling,  100  c.c.  of  the  clear  urine,  and  boil  for  a 
few  minutes  to  insure  complete  precipitation.  Filter 
through  a  weighed  filter,  wash  with  hot  water  until  free 
from  acid ;  dry,  and  weigh. 

Esbacli s  Method.  Fill  the  albuminometer  tube  to 
the  letter  U  with  the  clear  urine,  and  then  to  the  letter 
R  with  the  test  solution.  (See  below.)  Close  the  end  of 
the  tube  with  a  rubber  stopper,  invert  several  times, 
and  allow  the  mixture  to  stand  twenty-four  hours. 
When  the  precipitate  has  completely  settled,  read  off 
on  the  graduation  on  the  tube  the  number  of  grammes 
of  ser-albumin  per  litre  of  urine.  The  percentage  is 
derived  from  this  by  moving  the  decimal  point  one 
figure  to  the  left ;  thus  4  grammes  per  litre  would  be 
0.4  per  cent,  of  albumin.     If  the  urine  contains  con- 


URINE.  47 

siderable  albumin,  the  urine  must  first  be  diluted  with 
one  or  two  volumes  of  water  before  testing,  and  the 
result  multiplied  by  two  or  three,  as  the  volume  is 
doubled  or  trebled. 

The  test  solution  is  prepared  by  dissolving  10  grammes 
picric  acid  and  20  grammes  citric  acid  in  1000  c.c.  dis- 
tilled water.  In  order  to  save  time  the  tube  may  be 
placed  in  a  centrifugal  machine,  and  the  albumin  pre- 
cipitated in  three  or  four  minutes. 

MUCIN. 

Mix  some  saliva  with  some  normal  urine  and  apply 
the  following  tests  : 

Experiment.  Dilute  some  of  this  urine  in  a  test  tube 
with  water,  and  acidify  with  an  excess  of  acetic  acid. 
A  precipitate  of  mucin  is  formed.  This  precipitate 
dissolves  on  the  addition  of  caustic  soda,  and  is  again 
precipitated  on  the  addition  of  an  excess  of  acetic 
acid. 

Experiment.  To  another  portion  add  some  acetic 
acid,  and  then  some  potassium  ferrocyanide.  No 
cloudiness  or  precipitate. 

Experiment.  Repeat  Heller's  test,  with  some  of  the 
urine  containing  mucin.  A  zone  or  ring  is  obtained, 
but  it  is  not  so  sharply  defined,  and  is  not  at  the  point 
of  contact  of  the  two  liquids. 

Experiment.  Treat  the  urine  with  an  equal  volume 
of  a  saturated  solution  of  common  salt,  and  then  add 
a  solution  of  tannic  acid,  prepared  by  dissolving  5 
grammes  tannic  acid  in  a  mixture  of  10  c.c.  25  per  cent, 
acetic  acid  and  240  c.c.  40-50  per  cent,  alcohol. 


48  LESSON    VIII. 

In  the  presence  of  the  smallest  amounts  of  mucin  a 
precipitate  is  formed. 

PEPTONES. 

Add  a  little  peptone  to  some  normal  jirine  and  use 
this  for  the  following  test  : 

Experiment.  Saturate  some  of  the  urine  with  am- 
monium sulphate  after  having  slightly  acidified  it  with 
acetic  acid.  This  precipitates  the  albumin,  globulin, 
and  albumoses,  leaving  the  peptones  in  solution.  Filter 
and  test  the  filtrate  for  peptones  by  the  Biuret  test, 
Tanret's  test,  and  with  picric  acid. 


Lesson  IX. 

CARBOHYDRATES. 

Dextrose.  Urine  containing  sugar  as  a  rule  has  a  high 
specific  gravity,  a  light  color,  and  is  large  in  amount, 
sometimes  three  or  four  times  the  normal  amount. 

Before  testing  the  urine  for  sugar  see  that  it  does 
not  contain  any  protein  substances,  otherwise  they 
must  first  be  removed  by  coagulation  with  heat  and 
acetic  acic  and  filtration.  Albumin  interferes  with  the 
tests  for  sugar. 

Experiment.  Moore  s  test.  Add  some  caustic  soda 
to  some  urine  in  a  test  tube,  and  apply  heat  to  the 
upper  part  of  the  liquid.  In  the  presence  of  sugar  the 
urine  becomes  yellowish-brown  in  color,  as  seen  by 
observing  the  difference  in  the  color  between  the 
heated  and  the  unheated  portions.  The  depth  of  color 
depends  upon  the  quantity  of  sugar  present. 

The  color  disappears  on  the  addition  of  a  few  drops 
of  nitric  acid,  and  develops  an  odor  of  burnt  sugar 
(caramel). 

This  test  is  best  applied  to  decolorized  urines. 

Bile  pigments  produce  a  brown  color  with  caustic 
soda  without  the  application  of  heat. 

Experiment.  Trommer  s  test.  Treat  some  of  the  urine 
with  about  one  fourth  its  volume  of  caustic  soda,  and 

49 


50  LESSON  IX. 

then,  while  shaking,  add  a  10  per  cent,  copper  sulphate 
solution  until  the  precipitate  formed  just  redissolves, 
and  a  deep  blue  solution  is  the  result.  Apply  a  gentle 
heat  to  the  upper  part  of  the  liquid  and  a  reduction  of 
yellow  hydrated  cuprous  oxide,  and  then  red  cuprous 
oxide  is  obtained.  It  should  not  be  boiled  too  long, 
neither  should  the  precipitate  of  earthy  phosphates  be 
mistaken  for  a  reduction  of  cuprous  oxide,  which  settles 
quickly  to  the  bottom  of  the  test  tube. 

Experiment.  Fehling's  test.  The  solution  as  sug- 
gested by  Fehling  does  not  keep,  hence  it  is  advisable 
to  make  two  solutions  and  keep  them  separated.  I. 
Dissolve  34.65  grammes  pure  copper  sulphate  in  1000 
c.  c.  water.  2.  Dissolve  173  grammes  Rochelle  salt  in 
350  c.  c.  water,  adding  600  c.  c.  of  a  caustic  soda  solu- 
tion of  a  specific  gravity  of  1. 12  and  dilute  to  1000  c.  c. 
with  water.  For  use  mix  equal  parts  of  the  above  so- 
lutions and  dilute  with  an  equal  volume  of  water. 

Glycerine  may  be  used  instead  of  the  Rochelle  salt. 

In  applying  this  solution  to  the  urine  first  fill  a  test 
tube  one  half  full  of  the  solution  and  boil  for  a  few 
seconds.  No  change  or  reduction  should  be  observed 
in  the  liquid.  Now  add  the  urine  drop  by  drop  and  if 
sugar  is  present  a  reduction  of  yellow  hydrated  cuprous 
oxide  and  then  a  red  cuprous  oxide  is  seen.  The  test 
should  not  be  boiled  after  the  addition  of  the  urine 
but  only  warmed. 

If  a  reduction  occurs  in  the  test  solution  alone  on 
boiling  it  must  be  discarded. 

Urines  containing  large  amounts  of  uric  acid,  cre- 
atinin,  hippuric  acid,  hypoxanthin,  allantoin,  alkaloids, 
glycuronic  acid,  etc.,  may  give  a  marked  reduction  with 


URINE.  5 1 

these  preceding  tests  and  may  lead  to  error.  After 
the  administration  of  certain  drugs  such  as  benzoic 
acid,  turpentine,  chloral,  glycerin,  salicylic  acid,  etc.,  a 
reducing  substance  called  glycuronic  acid  is  eliminated 
in  the  urine,  which  may  mislead  the  experimenter. 
Urines  containing  an  excess  of  ammonia,  should  be 
boiled,  to  drive  off  the  ammonia,  before  applying  the 
above  tests  as  the  ammonia  has  a  tendency  to  dissolve 
any  cuprous  oxide  precipitated. 

Experiment.  Boettger  s  test.  Make  some  of  the 
urine  alkaline  with  sodium  carbonate  and  add  a  little 
solid  bismuth  sub-nitrate.  Heat  to  boiling  and  in  the 
presence  of  sugar  the  white  bismuth  sub-nitrate  will 
turn  gray,  brown  or  black,  depending  upon  the  amount 
of  sugar  present.  The  sugar  reduces  the  bismuth  sub- 
nitrate  into  bismuth  oxide  and  metalic  bismuth  (?). 

Experiment.  Nylander  s  modification  of  Boettger's 
test.  Dissolve  4  grammes  Rochelle  salt  in  a  solution 
of  10.33  grammes  caustic  soda  in  100  c.  c.  water.  Add 
to  this  2  grammes  bismuth  sub-nitrate  and  digest  on 
the  water  bath  until  as  much  of  the  bismuth  salt  has 
dissolved  as  possible. 

Heat  10  vols,  of  the  urine  with  1  vol.  of  the  above 
solution  for  a  few  minutes  when  a  black  precipitate  or 
a  dark  coloration  is  the  result. 

Before  applying  either  of  the  two  preceding  tests 
any  albumin  present  must  first  be  removed,  otherwise 
the  sulphur  of  the  albumin  will  turn  the  bismuth  salt 
black. 

Bismuth  sub-nitrate  is  not  reduced  by  the  substances 
mentioned  in  connection  with  Trommer's  or  Fehling's 
test. 


52  LESSON  IX. 

Experiment,  v.  Jakscti  s  test.  Place  25  c.  c.  of  the 
urine  in  a  small  flask  and  add  I  gramme  (15  grains) 
phenyl-hydrazine  hydrochloride  and  0.75  grammes  (10 
grains)  sodium  acetate.  Warm  on  the  water  bath  for 
at  least  one  hour.  Allow  to  cool  and  the  presence  of 
sugar  is  shown  by  the  presence  of  a  yellowish  deposit, 
which  may  appear  amorphous  to  the  naked  eye  but 
which,  when  examined  under  the  microscope,  is  seen  to 
contain  fine,  bright  yellow  needle-like  crystals,  either 
single  or  in  stars.  These  crystals  of  phenyl-glucosazone 
melt  at  2040  C,  and  are  highly  characteristic  and  cannot 
be  mistaken  for  any  other  substance. 

This  test  does  not  give  any  reaction  with  uric  acid, 
creatinin  hippuric  acid,  or  any  of  the  other  reducing 
substances  mentioned  above.  It  is  one  of  the  most 
delicate  and  most  reliable  of  all  known  tests  for  sugar 
in  the  urine. 

Experiment.  Make  some  of  the  urine  alkaline  with 
caustic  soda,  add  some  picric  acid  solution  and  heat 
the  upper  part  of  the  liquid.  In  the  presence  of  sugar 
a  dark  mahogany-red  color  will  be  produced.  This  test 
has  its  interferences,  namely  creatinin  gives  a  red 
coloration  with  this  test. 

Experiment.  Fermentation  test.  Shake  a  little  piece 
of  yeast  with  some  of  the  urine  and  fill  this  into  an 
Einhorn  Saccharometer  and  place  this  in  a  warm  lo- 
cality (400  C.)  for  12  hours.  The  dextrose  will  fer- 
ment with  the  evolution  of  carbon  dioxide,  which  will 
collect  in  the  tube  of  the  instrument. 

This  test  may  also  be  performed  by  simply  shaking 
some  yeast  with  four  ounces  of  the  urine  in  a  bottle 
which  is  tightly  corked  and  allow  it  to  stand  in  a  warm 


URINE.  53 

place  for  12  hours.  If  sugar  is  present,  on  removing 
the  cork  partly,  it  will  be  forcibly  ejected  and  the  car- 
bon dioxide  will  escape  as  a  froth.  If  much  sugar  is 
present  the  bottle  may  burst. 

The  fermentation  test  is  the  most  reliable  test  for 
dextrose  in  the  urine  and  is  readily  performed.  If  a 
urine  responds  to  the  reduction  tests  and  does  not  fer- 
ment with  yeast,  it  is  safe  to  conclude  that  no  dextrose 
is  present,  but  contains  other  substances  which  have  a 
reducing  action.  (Lactose  gives  the  reduction  tests  but 
does  not  ferment.) 

Quantitative  Estimation.  Experiment.  Robert's  diff- 
erential desity  method.  Take  the  specific  gravity  of 
the  urine  at  a  known  temperature,  and  then  add  a  little 
yeast  and  allow  it  to  ferment  in  a  warm  place  for  24 
hours.  Take  the  specific  gravity  again  at  the  same 
temperature  as  before.  Each  degree  of  specific  gravity 
lost  represents  1  grain  of  dextrose  per  fluid  ounce  of 
the  urine,  or  the  percentage  may  be  ascertained  by 
multiplying  each  degree  of  specific  gravity  lost  by  0.23. 
In  taking  the  specific  gravity,  be  careful  not  to  have 
any  gas-bubbles  attach  themselves  to  the  urinometer  ; 
otherwise  an  incorrect  reading  will  be  taken. 

Einhorns  Saccharometer.  Fill  one  of  the  instruments 
with  some  of  the  urine  to  be  tested  containing  a  little 
yeast,  and  the  second  instrument  with  normal  urine 
containing  the  same  amount  of  yeast.  Place  the  two 
side  by  side  in  a  warm  place  for  twelve  hours.  Read 
off  on  the  instrument  the  percentage  of  sugar  from  the 
volume  of  gas  collected.  The  object  of  the  second 
instrument  is  to  see  if  the  yeast  itself  gives  off  any  gas. 
These  results  are  only  approximate,  as  no  account  is 
taken  of  the  gas  dissolved  by  the  urine  itself. 


54  LESSON  IX. 

Fehlings  Titration  Method.  Place  20  c.  c.  of  the 
standard  Fehling's  solution  (prepared  as  above  de- 
scribed) in  a  flask,  and  dilute  with  two  volumes  water. 
Heat  to  boiling  and  add  small  amounts  of  the  urine 
from  a  burette  until  the  blue  color  of  the  solution  has  en- 
tirely disappeared.  After  each  addition  of  urine  from 
the  burette  it  is  advisable  to  boil  the  contents  of  the 
flask  and  allow  it  to  stand  a  few  seconds  so  that  the 
reduced  cuprous  oxide  may  settle,  and  so  the  observer 
may  see  if  the  blue  color  has  entirely  disappeared. 
When  the  blue  color  has  disappeared,  read  off  the 
number  of  cubic  centimeters  of  urine  used  from  the 
burette.  Since  it  requires  0.05  grammes  dextrose  to 
remove  the  blue  color  (namely  all  the  copper)  from  20 
c.  c.  of  Fehling's  solution,  therefore  the  quantity  of 
urine  used  must  have  contained  0.05  grammes  dextrose. 
From  this  calculate  the  percentage  of  dextrose  in  the 
urine. 

This  operation  should  be  repeated  to  get  exact  re- 
sults. If  the  original  urine  contains  a  large  amount  of 
dextrose,  then  dilute  it  first  with  a  known  volume  of 
water. 

Polariscope.  Dextrose  has  a  right-handed  or  dextro- 
rotatory action  on  polarized  light.  Its  specific  rotatory 
power  is  (a)D  = +  52.6.  In  using  this  instrument  for  the 
determination  of  the  amount  of  sugar  in  a  urine,  first 
decolorize  the  urine  by  shaking  it  with  freshly  burnt 
animal  charcoal,  filter  and  place  the  perfectly  decolor- 
ized and  clear  urine  in  the  glass  tube  provided  for  the 
purpose.  Place  this  later  between  the  polarizer  and 
the  analyser,  and  rotate  the  analyser  until  both  halves 
of  the  field  of  view  are  equally  illuminated.     Read  off 


URINE.  5  5 

on  the  scale  by  means  of  the  vernir  the  percentage  of 
dextrose  in  the  urine. 

Urines  may  also  contain  : 

Lactose,  in  the  urine  of  women  when  nursing. 

Inosite. 

Levulose,  occasionally  in  diabetic  urines. 

Maltose. 

BILE. 

Urine  containing  bile  pigments  has  a  color  varying 
from  bright  yellow  to  a  greenish-brown,  and  when 
shaken,  readily  gives  a  foam  having  a  yellow  color. 

It  is  generally  ropy,  with  a  high  specific  gravity. 

Experiment.  Apply  Gmelin's  test  to  the  urine  as 
described  in  Lesson  5,  Experiment  20.  The  test  should 
be  allowed  to  stand  some  little  time  so  as  to  allow  the 
characteristic  rings  of  color  to  develop. 

Experiment.  Apply  Huppert's  test,  as  described  in 
Lesson  5,  Experiment  21,  to  the  urine. 

Experiment.  Repeat  Smith's  test  as  described  in 
Lesson  5,  Experiment  22. 

The  bile  acids  can  only  be  tested  for  in  a  urine  with 
great  difficulty. 

BLOOD. 

Blood  occurs  in  urines  in  cases  of  hematuria  where 
we  have  the  presence  of  blood  corpuscles,  and  in  cases 
of  hemoglobinuria  where  we  find  the  blood  pigments 
with  very  few  corpuscles.  The  presence  of  blood  in  a 
sample  of  urine  may  be  seen  by  its  characteristic  color 
and  its  smoky  and  dichroic  appearance.  The  presence 
of  blood  corpuscles  is  determined  by  microscopic 
examination. 


$6  LESSON  IX. 

Experiment.  Test  the  presence  of  albumin  in  the 
urine  containing  blood. 

Experiment.  Make  some  of  the  urine  alkaline  with 
caustic  soda  and  boil  so  as  to  precipitate  the  earthy- 
phosphates.  They  will  be  colored  bright  red,  due  to 
their  retaining  blood  pigments.  Collect  this  precipi- 
tate on  a  filter  and  use  it  for  the  obtainment  of  Haemin 
or  Teichmann's  crystals,  as  described  in  Lesson  5, 
Experiment  16.  In  this  case  there  is  no  need  of  add- 
ing any  common  salt. 

Experiment.  Repeat  Experiment  17,  Lesson  5,  with 
the  urine,  but  instead  of  using  hydrogen  peroxide,  use 
ether  which  has  been  ozonized  (by  shaking  it  with 
hydrogen  peroxide).  As  the  ether  floats  on  the  mix- 
ture of  urine  and  tincture  of  guaiacum  it  forms  a  blue 
ring  in  the  presence  of  blood. 

Experiment.  Slightly  acidify  the  urine  with  acetic 
acid,  filter  and  place  it  in  front  of  the  slit  of  the  spectro- 
scope. Two  absorption  bands  will  be  seen,  as  described 
in  Lesson  5,  Experiment  14. 

PUS. 

Urine  containing  pus  corpuscles  has  a  cloudy  appear- 
ance and  is  clarified  by  filtration  with  difficulty. 

Microscopical  examination  reveals  the  presence  of 
pus  corpuscles. 

Experiment.  Test  for  albumin  in  the  urine  contain- 
ing pus.     It  will  be  present  in  large  amounts. 

Experiment.  Allow  the  corpuscles  to  settle  or  facili- 
tate by  means  of  a  centrifugal  machine  and  treat  them, 
after  decanting  the  urine  from  them,  with  a  small  piece 
of  caustic  soda  and  stir.     After  a  moment  or  two  the 


URINE.  57 

corpuscles  are  converted  into  a  ropy  and  gelatinous 
mass. 

Experiment.  Acidify  the  urine  with  acetic  acid,  filter 
and  treat  a  portion  of  the  contents  of  the  filter  with  a 
few  drops  of  tincture  of  guaiacum  (which  has  been  kept 
in  the  dark),  when  in  the  presence  of  pus  the  filter 
paper  is  colored  a  deep  blue. 

Experiment.  Place  some  of  the  contents  of  the  filter 
obtained  above  on  a  slide  and  treat  it  with  a  drop  of  a 
solution  of  iodine  in  water  containing  some  potassium 
iodide.  In  the  presence  of  pus  a  dark,  mahogany- 
brown  color,  due  to  the  glycogen  contained  therein,  is 
the  result.     Epithelium  cells  do  not  give  this  color. 

UROBILIN. 

This  is  the  normal  coloring  matter  of  the  urine.  It 
is  markedly  increased  in  fevers. 

Experiment.  Make  some  of  the  urine  alkaline  with  am- 
monium hydrate,  and  filter  off  the  precipitate  of  earthy 
phosphates.  To  the  filtrate  add  a  few  drops  of  a  solu- 
tion of  zinc  chloride,  which  produces  a  beautiful  green- 
ish fluorescence  by  reflected  light.  The  above  solution 
exhibits,  with  the  spectroscope,  a  dark  absorption  band 
between  the  Fraunhofer's  lines  b  and  F. 

INDICAN. 

This  substance  is  derived  from  the  indol,  produced 
in  the  intestine  by  putrefactive  processes,  which  is 
oxidized  into  indoxyl  in  the  blood  ;  this,  entering  in 
combination  with  sulphuric  acid,  is  eliminated  in  the 
urine  as  sodium  or  potassium  indoxyl  sulphate  or  indi- 
can,  (KO.C8H8N.O.S08). 


58  LESSON  IX. 

Experiment.  Treat  a  small  amount  of  the  urine  in  a 
test  tube  with  an  equal  volume  of  hydrochloric  acid 
and  two  or  three  drops  sodium  hypochlorite  solution, 
and  then  a  small  amount  of  chloroform.  Shake  and 
put  aside  for  a  little  while.  The  indigo  set  free  from 
the  indican  is  taken  up  by  the  chloroform,  coloring  it 
blue  to  a  greater  or  less  extent,  depending  upon  the 
amount  of  indican  present  in  the  urine. 

Experiment.  Boil  equal  volumes  of  the  urine  and 
hydrochloric  acid,  containing  a  few  drops  nitric  acid  ; 
allow  to  cool  and  shake  with  a  little  chloroform.  The 
chloroform  is  colored  violet,  due  to  the  setting  free  of 
indigo  blue  and  indigo  red  from  the  indican. 


Lesson  X. 

SEDIMENTS  (Chemical  or  unorganized). 

From  acid  urines.     From  neutral  or  slightly  alkaline 

(fixed)  urines. 
Uric  acid.  Sodium  urate. 

Sodium  urate.  Calcium  oxalate. 

Calcium  oxalate.  Calcium  carbonate. 

Acid  calcium  carbonate.    Di-calcium  phosphate. 

Tri-calcium  phosphate. 
Ammonium-magnesium  phos- 
phate. 
From  ammoniacal  urines. 
Ammonium  urate. 
Ammonium-magnesium  phosphate. 
Tri-calcium  phosphate. 
Calcium  carbonate. 

In  testing  the  sediments  the  urine  should  be  allowed 
to  stand  in  a  conical  vessel  in  a  cool  place  until  all  the 
sediment  has  settled  to  the  bottom.  It  may  be  col- 
lected by  decantation,  or  better,  by  means  of  a  pipette. 
Hold  the  first  finger  over  the  end  of  the  pipette  and 
introduce  it  to  the  bottom  of  the  vessel  into  the  sedi- 
ment. Now  remove  the  finger  slowly  and  the  sediment 
will  rise  in  the  tube.  Close  the  end  of  the  tube  tightly 
again  with  the  finger  and  remove  it  from  the  vessel. 
Allow  the  contents  of  the  pipette  to  flow  on  a  few 

59 


60  LESSON  X. 

pieces  of  filter  paper,  which  absorbs  the  urine,  leaving 
the  sediment  on  the  paper,  which  may  be  removed  by 
the  aid  of  a  knife  or  spatula,  and  tested  as  described 
below. 

The  sediment  may  also  be  obtained  readily  by  means 
of  a  centrifugal  machine. 

URIC   ACID,    C5H4N403. 

Generally  colored  red. 

Experiment.  Heat  some  on  a  piece  of  platinum  foil. 
It  blackens  and  burns  up  entirely,  leaving  no  residue. 

Experiment.  Place  a  little  in  water  in  a  test  tube. 
It  does  not  dissolve.  When  a  drop  or  two  of  sodium 
carbonate  is  added  it  dissolves  readily. 

Experiment.  Apply  the  Murexid  test  to  a  portion 
of  the  sediment.  A  purple  coloration  shows  the  pres- 
ence of  uric  acid.     (See  uric  acid.) 

Experiment.  Repeat  Schiff's  test  with  the  solution 
obtained  above.  A  black  reduction  denotes  the  pres- 
ence of  uric  acid.     (See  uric  acid.) 

SODIUM   URATE,    Na3C5H3N403. 

Experiment.  Heat  a  little  on  the  foil.  It  blackens 
and  burns  with  a  yellow  color  to  the  flame,  if  looked  at 
through  a  piece  of  blue  glass.  It  does  not  burn  up  en- 
tirely, but  leaves  a  residue,  which  if  placed  on  a  piece 
of  moistened  red  litmus  paper  turns  it  blue,  due  to  the 
sodium  carbonate  in  the  residue. 

Experiment.  Place  a  little  in  water.  It  does  not 
dissolve,  but  dissolves  readily  when  a  drop  of  sodium 
carbonate  solution  is  added. 

Experiment.  Repeat  the  Murexid  test  with  some  of 
the  sediment. 


SEDIMENTS.  6 1 

Experiment.  Repeat  Schiff' s  test  with  the  solution 
of  the  sediment  obtained  above.  A  black  reduction  is 
the  result. 

CALCIUM  OXALATE,  CaC204. 

Heat  some  on  the  foil.  It  blackens  slightly  but 
does  not  burn  or  disappear.  Place  this  residue  in  a 
watch  glass  and  add  a  little  water  and  a  drop  of  acetic 
acid.  It  effervesces,  due  to  the  discharge  of  carbon 
dioxide,  and  dissolves.     Retain  this  solution. 

The  application  of  heat  to  the  sediment  has  con- 
verted the  calcium  oxalate  into  calcium  carbonate, 
which  effervesces  with  an  acid.    CaC304  =  CaC03  +  CO. 

Experiment.  Heat  another  portion  of  the  sediment 
for  a  few  minutes  in  the  flame.  Place  a  particle  on  a 
piece  of  moistened  red  litmus  paper.  It  turns  it  blue, 
showing  that  calcium  oxide  has  been  formed  by  the 
protracted  heating  of  the  calcium  oxalate.  CaC204  = 
CaO  +  CO  +  C03. 

Place  the  remainder  of  the  residue  in  some  water  in 
a  test  tube  or  watch  glass.  It  dissolves,  especially  on 
the  addition  of  a  drop  of  acetic  acid. 

Experiment.  Add  some  ammonium  oxalate  to  some 
of  the  solution  obtained  above.  A  white  precipitate 
of  calcium  oxalate  shows  the  presence  of  calcium. 

Calcium  oxalate  does  not  give  the  Murexid  test. 

ACID  CALCIUM  PHOSPHATE,  Ca  (H2P04)3. 

Experiment.  Heat  some  on  the  foil.  It  does  not 
blacken  nor  does  it  burn  up.     No  apparent  change. 

Experiment.  Place  some  in  a  test  tube  with  water. 
Add  some  acetic  acid  and  apply  heat  if  necessary.     It 


62  LESSON  X. 

dissolves.  Divide  the  solution  in  two  portions.  To 
one  portion  add  a  solution  of  ammonium  oxalate.  A 
white  precipitate  of  calcium  oxalate  shows  the  presence 
of  calcium. 

To  the  other  portion  add  a  solution  of  uranium 
acetate.  A  white  precipitate  of  uranium  phosphate 
denotes  the  presence  of  phosphoric  acid. 

Acid  calcium  phosphate  does  not  give  the  Murexid 
test. 

Di-calcium  phosphate,  CaHP04. 

Tri-calcium  phosphate,  Ca3  (P04)3. 

Occurring  in  neutral  or  alkaline  urines  are  tested  for 
as  described  under  acid  calcium  phosphate.  Their  mi- 
croscopic appearances  are  different. 

CALCIUM  CARBONATE,  CaC03. 

Experiment.  Heat  some  on  the  foil.  It  does  not 
blacken  nor  burn  up.  No  apparent  change.  Place  a 
particle  of  the  heated  residue  on  a  piece  of  moistened 
red  litmus  paper.  It  turns  blue,  showing  the  presence 
of  calcium  oxide,  formed  by  heating  the  carbonate. 
CaCOs  =  CaO  +  COs. 

Experiment.  Place  some  of  the  sediment  in  a  test 
tube  with  water.  It  does  not  dissolve,  but  readily  dis- 
solves on  the  addition  of  a  drop  or  two  of  acetic  acid 
with  an  effervescence  showing  that  it  is  a  carbonate  by 
the  escape  of  carbon  dioxide  gas. 

Experiment.  Add  some  ammonium  oxalate  to  the 
solution  obtained  in  the  previous  experiment.  A  white 
precipitate  of  calcium  oxalate  shows  the  presence  of 
calcium. 

This  sediment  does  not  give  the  Murexid  test. 


SEDIMENTS.  63 

AMMONIUM-MAGNESIUM   PHOSPHATE  OR  TRIPLE  PHOS- 
PHATE, NH4,  MG,  P04. 

Heat  on  the  piece  of  foil.  It  does  not  blacken,  but 
gives  off  an  odor  of  ammonia,  which  gives  dense  white 
fumes  with  a  drop  of  hydrochloric  acid  on  a  glass  rod. 

A  residue  of  magnesium  pyro-phosphate  is  left  on 
the  foil. 

Experiment.  Place  some  of  the  sediment  in  a  test 
tube  and  add  some  caustic  soda.  A  marked  odor  of 
ammonia  is  evolved,  and  a  piece  of  moistened  red  lit- 
mus paper  is  turned  blue  by  the  vapors,  if  held  at  the 
mouth  of  the  test  tube. 

Experiment.  Place  some  of  the  sediment  with  water 
in  a  test  tube  and  add  a  few  drops  acetic  acid.  It  dis- 
solves without  effervescence. 

Experiment.  Add  some  uranium  acetate  to  some  of 
the  above  solution.  A  white  precipitate  of  uranium 
phosphate  denotes  the  presence  of  phosphoric  acid. 

It  does  not  respond  to  the  Murexid  test. 

AMMONIUM   URATE,  (N2H4)C5H3N403. 

Experiment.  Heat  some  on  the  foil.  It  blackens 
and  burns  up,  leaving  no  residue. 

Experiment.  Place  some  of  the  sediment  on  a  watch 
glass  and  add  a  few  drops  caustic  soda.  Apply  a 
gentle  heat  to  the  contents  of  the  watch  glass,  and 
hold  a  piece  of  moistened  red  litmus  directly  over  the 
watch  glass.  It  turns  blue,  showing  that  ammonia  has 
been  evolved  by  the  action  of  the  caustic  soda  on  the 
ammonium  urate. 

Experiment.     Place  some  of  the  sediment  in  water  in 


64  LESSON  X. 

a  test  tube.  It  does  not  dissolve,  but  readily  dissolves 
on  the  addition  of  a  few  drops  sodium  carbonate. 

Experiment.  Apply  Schiff's  test  to  the  above  solu- 
tion. A  black  reduction  shows  the  presence  of  uric 
acid. 

Experiment.  Apply  the  Murexid  test  to  some  of  the 
sediment  on  a  watch  glass.  A  purple  coloration  de- 
notes the  presence  of  uric  acid. 

Cystin,  leucin,  tyrosin,  and  xanthin  have  been  ob- 
served in  sediments  in  very  rare  cases. 

CALCULI. 

Calculi  may  consist  of  only  one  ingredient,  but  more 
frequently  two  or  more  deposits  occur  in  separate 
layers.  Most  calculi  have  a  nucleus  generally  of  uric 
acid. 

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a  piece  of  writing  paper  and  cut  through  the  centre  by 
means  of  a  fine  saw.  Each  layer  should  be  examined 
separately  by  the  following  scheme  : 


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